Anti-claudin 18 antibodies and methods of use thereof

ABSTRACT

Antibodies that specifically bind to the human tight junction molecule CLDN18.2 and have functional properties that make them suitable for use in antibody-based immunotherapies of a disease associated with aberrant expression of CLDN18.2 are disclosed.

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No.62/800,359, filed on Feb. 1, 2019, and U. S. Provisional Application No.62/891,925 filed on Aug. 26, 2019. The entire teachings of the aboveapplications are incorporated herein by reference.

FIELD OF THE DISCLOSURE

The present disclosure generally relates to claudin 18.2(CLDN18.2)-specific antibodies and antibody fragments thereof. Theantibodies are useful for immunotherapy of a disease associated withaberrant expression of CLDN18.2, including epithelial cell-derivedprimary and metastatic cancer.

BACKGROUND OF THE INVENTION

Two alternatively spliced human claudin 18 transcript variants, encodingdistinct isoforms that exhibit lung-restricted (CLDN18.1) andstomach-restricted (CLDN18.2) expression (Niimi et al., Mol. Cell. Biol.21:7380-90, 2001), in a promoter-dependent manner, have previously beendescribed. The primary protein sequences of the splice variants differin the N-terminal portion that comprises the N-terminal intracellularregion, first transmembrane region (TMD1), and extracellular loop one(ECL1). CLDN18.2 is one of a few members of the human claudin familywith strict restriction to one cell lineage (Türeci et al.). Morespecifically, it provides a highly selective gastric lineage (e.g.,gastrocyte-specific) marker with an expression pattern that isrestricted to short-lived differentiated epithelial cells and absentfrom the stem cell zone of gastric glands (Sahin et al., Clin. CancerRes. 14 (23) 7624-7634, 2008).

CLDN18.2 is retained in malignant transformation and is expressed in asignificant portion of primary tumors and their metastasis. Sahin et al.also reported that CLDN18.2, but not CLDN18.1, is frequentlyoverexpressed in several different types of cancers, includingpancreatic, stomach, esophageal, lung, and ovarian cancers. Therefore,in the context of cancer, CLDN18.2 does not remain restricted to thegastric cell lineage (Sahin et al.) . Considered together, the findingsof published reports establish that CLDN18.2 provides both a diagnostictool and a druggable target for the development of cancerimmunotherapies of diseases associated with epithelial cell-derivedtumors.

It has been reported that tight junction permeability is often higher intumor tissues than in normal tissues, leading to the speculation thatclaudin proteins on tumor cells may be more accessible than in normaltissues with intact tight junctions. This possibility makes claudinproteins attractive targets for therapeutic cancer interventions. Inaddition, published expression profiling results suggest that cancertherapies targeting CLDN18.2 will have favorable systemic toxicityprofiles because normal turnover and homeostasis processes replenishgastrointestinal epithelial cells every two to seven days (Sahin etal.). Transient gastrointestinal toxicity of limited duration is acommon and manageable adverse event for cancer immunotherapeutics.

Pancreatic and gastroesophageal cancers are among the malignancies withthe highest unmet medical need (Sahin, et al.). Despite the fact thatgastric cancer and pancreatic cancer contribute to significantcancer-related morbidity and mortality, the treatment options arelimited. Thus, the need exists for anti-CLDN18.2 specific antibodies andbinding agents for use in the immunotherapy of cancer associated withepithelial cell-derived primary and metastatic solid tumors.

SUMMARY OF THE INVENTION

The present disclosure addresses the above need by providing antibodiesand antibody fragments that specifically bind to the human tightjunction molecule CLDN18 isoform 2 (CLDN18.2) and have desirablefunctional properties. The anti-CLDN18.2 antibodies or antibodyfragments thereof, or bispecific molecules, or fusion proteinscomprising the CLDN18.2 antibodies or antibody fragments may be used forantibody-based therapies of diseases associated with dysregulation ofCLDN18.2 expression. For example, the antibodies may be used fortreating solid tumor cancer diseases associated with cells expressingCLDN18.2, such as gastric, pancreatic and esophageal, lung, ovarian,colon, and hepatic cancers.

In some embodiments, the antibody of the invention may be a monoclonal,chimeric, humanized or human antibody, a component of a bispecific ormultispecific antibody, or an antigen-binding portion of an antibody,which binds to the first extracellular domain/loop of CLDN18.2 andexhibits one or more of the following properties: (a) specificity forCLDN18.2 (e.g. binding to human CLDN18.2 but not to CLDN18.1); (b)ability to mediate ADCC killing of cells expressing CLDN18.2; and (c)efficiently internalized upon CLDN18.2 binding on CLDN18.2 expressingcells and therefore suitable for ADC development.

In some embodiments, the anti-CLDN18.2 specific antibodies or antibodyfragments thereof bind preferentially to CLDN18.2 (isoform 2) naturallyexpressed by human tumor cells and do not bind to CLDN18 isoform 1(CLDN18.1). As a consequence of binding CLDN18.2 expressed on thesurface of a target cell, the disclosed antibodies can mediate targetcell killing by one or more mechanisms of action, such as induction ofapoptosis, inhibition of proliferation, CDC lysis, ADCC lysis ordelivery of a cytotoxic agent. In one embodiment, the target cells areprimary or metastatic cancer cells.

In some embodiments, the anti-CLDN18.2 antibody is a full-lengthantibody.

In some embodiments, the anti-CLDN18.2 antibody is an antibody fragment.In further embodiments, the antibody fragment is selected from the groupconsisting of: Fab, Fab′, F(ab)2, Fd, Fv, scFv, and scFv-Fc fragment, asingle-chain antibody, a minibody, and a diabody.

In some embodiments, the anti-CLDN18.2 antibody is a monoclonalantibody.

In some embodiments, the anti-CLDN18.2 antibody is a murine antibody.

In some embodiments, the anti-CLDN18.2 antibody is a human antibody. Insome embodiments, the anti-CLDN18.2 antibody is a humanized antibody.

In some embodiments, the anti-CLDN18.2 antibody is a chimeric antibody.

In some embodiments, the anti-CLDN18.2 antibody is a bispecific antibodycomprising either an unmodified (e.g., naturally occurring) Fc fragmentor a modified Fc fragment designed to optimize, or in the alternative toeliminate, particular Fc-mediated functions.

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 47(HCDR1), SEQ ID NO: 48 (HCDR2), and SEQ ID NO: 49 (HCDR3); and/or alight chain variable region having SEQ ID NO: 50 (LCDR1), SEQ ID NO: 51(LCDR2), and SEQ ID NO: 52 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 53(HCDR1), SEQ ID NO: 54 (HCDR2), and SEQ ID NO: 55 (HCDR3); and/or alight chain variable region having SEQ ID NO: 56(LCDR1), SEQ ID NO: 57(LCDR2), and SEQ ID NO: 58 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 59(HCDR1), SEQ ID NO: 60 (HCDR2), and SEQ ID NO: 61 (HCDR3); and/or alight chain variable region having SEQ ID NO: 62 (LCDR1), SEQ ID NO: 63(LCDR2), and SEQ ID NO: 64 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 65(HCDR1), SEQ ID NO: 66 (HCDR2), and SEQ ID NO: 67 (HCDR3); and/or alight chain variable region having SEQ ID NO:68 (LCDR1), SEQ ID NO: 69(LCDR2), and SEQ ID NO:70 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 71(HCDR1), SEQ ID NO: 72 (HCDR2), and SEQ ID NO: 73 (HCDR3); and/or alight chain variable region having SEQ ID NO: 74 (LCDR1), SEQ ID NO: 75(LCDR2), and SEQ ID NO: 76 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 77(HCDR1), SEQ ID NO: 78 (HCDR2), and SEQ ID NO: 79 (HCDR3); and/or alight chain variable region having SEQ ID NO:80 (LCDR1), SEQ ID NO: 81(LCDR2), and SEQ ID NO: 82 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 83(HCDR1), SEQ ID NO: 84 (HCDR2), and SEQ ID NO: 85 (HCDR3); and/or alight chain variable region having SEQ ID NO: 86 (LCDR1), SEQ ID NO: 87(LCDR2), and SEQ ID NO: 88 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 89(HCDR1), SEQ ID NO: 90 (HCDR2), and SEQ ID NO: 91 (HCDR3); and/or alight chain variable region having SEQ ID NO: 92 (LCDR1), SEQ ID NO: 93(LCDR2), and SEQ ID NO: 94 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 95(HCDR1), SEQ ID NO: 96 (HCDR2), and SEQ ID NO: 97 (HCDR3); and/or alight chain variable region having SEQ ID NO: 98 (LCDR1), SEQ ID NO: 99(LCDR2), and SEQ ID NO: 100 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 101(HCDR1), SEQ ID NO: 102 (HCDR2), and SEQ ID NO: 103 (HCDR3); and/or alight chain variable region having SEQ ID NO: 104 (LCDR1), SEQ ID NO:105 (LCDR2), and SEQ ID NO: 106 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 107(HCDR1), SEQ ID NO: 108 (HCDR2), and SEQ ID NO: 109 (HCDR3); and/or alight chain variable region having SEQ ID NO:110 (LCDR1), SEQ ID NO: 111(LCDR2), and SEQ ID NO: 112 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 113(HCDR1), SEQ ID NO: 114 (HCDR2), and SEQ ID NO: 115 (HCDR3); and/or alight chain variable region having SEQ ID NO: 116 (LCDR1), SEQ ID NO:117 (LCDR2), and SEQ ID NO: 118 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 119(HCDR1), SEQ ID NO: 120 (HCDR2), and SEQ ID NO: 121 (HCDR3); and/or alight chain variable region having SEQ ID NO: 122 (LCDR1), SEQ ID NO:123 (LCDR2), and SEQ ID NO: 124 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 125(HCDR1), SEQ ID NO: 126 (HCDR2), and SEQ ID NO: 127 (HCDR3); and/or alight chain variable region having SEQ ID NO: 128 (LCDR1), SEQ ID NO:129 (LCDR2), and SEQ ID NO: 130 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 131(HCDR1), SEQ ID NO: 132 (HCDR2), and SEQ ID NO: 133 (HCDR3); and/or alight chain variable region having SEQ ID NO: 134 (LCDR1), SEQ ID NO:135 (LCDR2), and SEQ ID NO: 136 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 137(HCDR1), SEQ ID NO: 138 (HCDR2), and SEQ ID NO:139 (HCDR3); and/or alight chain variable region having SEQ ID NO:140 (LCDR1), SEQ ID NO:141(LCDR2), and SEQ ID NO: 142 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 143(HCDR1), SEQ ID NO: 144 (HCDR2), and SEQ ID NO: 145 (HCDR3); and/or alight chain variable region having SEQ ID NO:146 (LCDR1), SEQ ID NO: 147(LCDR2), and SEQ ID NO: 148 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 149(HCDR1), SEQ ID NO: 150 (HCDR2), and SEQ ID NO: 151 (HCDR3); and/or alight chain variable region having SEQ ID NO: 152 (LCDR1), SEQ ID NO:153 (LCDR2), and SEQ ID NO: 154 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 155(HCDR1), SEQ ID NO: 156 (HCDR2), and SEQ ID NO: 157 (HCDR3); and/or alight chain variable region having SEQ ID NO: 158 (LCDR1), SEQ ID NO:159 (LCDR2), and SEQ ID NO: 160 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO:161(HCDR1), SEQ ID NO: 162 (HCDR2), and SEQ ID NO: 163 (HCDR3); and/or alight chain variable region having SEQ ID NO: 164 (LCDR1), SEQ ID NO:165 (LCDR2), and SEQ ID NO: 166 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 167(HCDR1), SEQ ID NO: 168 (HCDR2), and SEQ ID NO: 169 (HCDR3); and/or alight chain variable region having SEQ ID NO: 170 (LCDR1), SEQ ID NO:171(LCDR2), and SEQ ID NO: 172 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO:173(HCDR1), SEQ ID NO: 174 (HCDR2), and SEQ ID NO: 175 (HCDR3); and/or alight chain variable region having SEQ ID NO: 176 (LCDR1), SEQ ID NO:177 (LCDR2), and SEQ ID NO: 178 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 179(HCDR1), SEQ ID NO: 180 (HCDR2), and SEQ ID NO:181 (HCDR3); and/or alight chain variable region having SEQ ID NO: 182 (LCDR1), SEQ ID NO:183 (LCDR2), and SEQ ID NO: 184 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 221(HCDR1), SEQ ID NO: 222 (HCDR2), and SEQ ID NO: 223 (HCDR3); and/or alight chain variable region having SEQ ID NO: 224 (LCDR1), SEQ ID NO:225 (LCDR2), and SEQ ID NO: 226 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 227(HCDR1), SEQ ID NO: 228 (HCDR2), and SEQ ID NO:229 (HCDR3); and/or alight chain variable region having SEQ ID NO: 230 (LCDR1), SEQ ID NO:231(LCDR2), and SEQ ID NO: 232 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 233(HCDR1), SEQ ID NO: 234 (HCDR2), and SEQ ID NO: 235 (HCDR3); and/or alight chain variable region having SEQ ID NO: 236 (LCDR1), SEQ ID NO:237 (LCDR2), and SEQ ID NO: 238 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 239(HCDR1), SEQ ID NO: 240 (HCDR2), and SEQ ID NO: 241 (HCDR3); and/or alight chain variable region having SEQ ID NO: 242 (LCDR1), SEQ ID NO:243 (LCDR2), and SEQ ID NO: 244 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 245(HCDR1), SEQ ID NO: 246 (HCDR2), and SEQ ID NO: 247 (HCDR3); and/or alight chain variable region having SEQ ID NO: 248 (LCDR1), SEQ ID NO:249 (LCDR2), and SEQ ID NO: 250 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 251(HCDR1), SEQ ID NO: 252 (HCDR2), and SEQ ID NO: 253 (HCDR3); and/or alight chain variable region having SEQ ID NO: 254 (LCDR1), SEQ ID NO:255 (LCDR2), and SEQ ID NO: 256 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 257(HCDR1), SEQ ID NO: 258 (HCDR2), and SEQ ID NO: 259 (HCDR3); and/or alight chain variable region having SEQ ID NO: 260 (LCDR1), SEQ ID NO:261 (LCDR2), and SEQ ID NO: 262 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 263(HCDR1), SEQ ID NO: 264 (HCDR2), and SEQ ID NO: 265 (HCDR3); and/or alight chain variable region having SEQ ID NO: 266 (LCDR1), SEQ ID NO:267 (LCDR2), and SEQ ID NO: 268 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 269(HCDR1), SEQ ID NO: 270 (HCDR2), and SEQ ID NO: 271 (HCDR3); and/or alight chain variable region having SEQ ID NO: 272 (LCDR1), SEQ ID NO:273 (LCDR2), and SEQ ID NO: 274 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 275(HCDR1), SEQ ID NO: 276 (HCDR2), and SEQ ID NO: 277 (HCDR3); and/or alight chain variable region having SEQ ID NO: 278 (LCDR1), SEQ ID NO:279 (LCDR2), and SEQ ID NO: 280 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 281(HCDR1), SEQ ID NO: 282 (HCDR2), and SEQ ID NO: 283 (HCDR3); and/or alight chain variable region having SEQ ID NO: 284 (LCDR1), SEQ ID NO:285 (LCDR2), and SEQ ID NO: 286 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 287(HCDR1), SEQ ID NO: 288 (HCDR2), and SEQ ID NO: 289 (HCDR3); and/or alight chain variable region having SEQ ID NO: 290 (LCDR1), SEQ ID NO:291 (LCDR2), and SEQ ID NO: 292 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 293(HCDR1), SEQ ID NO: 294 (HCDR2), and SEQ ID NO: 295 (HCDR3); and/or alight chain variable region having SEQ ID NO: 296 (LCDR1), SEQ ID NO:297 (LCDR2), and SEQ ID NO: 298 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 299(HCDR1), SEQ ID NO: 300 (HCDR2), and SEQ ID NO: 301 (HCDR3); and/or alight chain variable region having SEQ ID NO: 302 (LCDR1), SEQ ID NO:303 (LCDR2), and SEQ ID NO: 304 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 311(HCDR1), SEQ ID NO: 312 (HCDR2), and SEQ ID NO: 313(HCDR3); and/or alight chain variable region having SEQ ID NO: 314 (LCDR1), SEQ ID NO:315 (LCDR2), and SEQ ID NO: 316 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 317(HCDR1), SEQ ID NO: 318 (HCDR2), and SEQ ID NO: 319 (HCDR3); and/or alight chain variable region having SEQ ID NO: 320 (LCDR1), SEQ ID NO:321 (LCDR2), and SEQ ID NO: 322 (LCDR3).

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a heavy chain variable region having SEQ ID NO: 323(HCDR1), SEQ ID NO: 324 (HCDR2), and SEQ ID NO: 325 (HCDR3); and/or alight chain variable region having SEQ ID NO: 326 (LCDR1), SEQ ID NO:327 (LCDR2), and SEQ ID NO: 328 (LCDR3).

In some embodiments, the anti-CLDN 18.2 antibodies or antibody fragmentsthereof comprise a variable heavy chain sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 215, 217, and 219.

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a variable light chain sequence selected from the groupconsisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 216, 218, and 220.

The present invention further provides anti-CLDN18.2 specific antibodiesor binding fragments thereof comprising an antibody or CLDN18.2 bindingfragment comprising a specific combination or pair of variable heavy andvariable light chain sequences.

In an embodiment, an anti-CLDN18.2 specific antibodies or bindingfragments thereof comprise an antibody or CLDN18.2 binding fragmentthereof comprising a variable heavy chain sequence selected from thegroup consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45, respectively, pairedwith a variable light chain sequence selected from the group consistingof SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, and 46, respectively.

In an embodiment, the anti-CLDN18.2 specific antibodies or bindingfragments thereof comprise an antibody or CLDN18.2 binding fragmentthereof comprising a variable heavy chain sequence selected from thegroup consisting of SEQ ID NOS: 185, 187, 189, 191, 193, 195, 197, 199,201, 203, 205, 207, 209, 211, 215, 217, and 219, respectively, pairedwith a and a variable light chain sequence selected from the groupconsisting of SEQ ID NOS: 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 216, 218, and 220, respectively.

In an embodiment, the anti-CLDN18.2 specific antibodies or bindingfragments thereof comprise an antibody or CLDN18.2 binding fragmentcomprising a specific combination or pair of variable heavy and variablelight chain sequences. In an alternative embodiment, the anti-CLDN18.2specific antibodies or binding fragments thereof comprise an antibody orCLDN18.2 binding fragment or binding agent comprising defined sets orcombinations of CDR sequences derived from the VH and/or VL domainregions of the anti-CLDN18.2 antibodies of the invention.

In some embodiments, the anti-CLDN18.2 antibody or antigen-bindingfragment thereof comprises a specific pair of variable heavy chain andvariable light chain sequences, selected from the followingcombinations.

-   (a) a variable heavy chain sequence comprising SEQ ID NO:1 and a    variable light chain sequence comprising SEQ ID NO:2;-   (b) a variable heavy chain sequence comprising SEQ ID NO:3 and a    variable light chain sequence comprising SEQ ID NO:4;-   (c) a variable heavy chain sequence comprising SEQ ID NO:5 and a    variable light chain sequence comprising SEQ ID NO:6;-   (d) a variable heavy chain sequence comprising SEQ ID NO:7 and a    variable light chain sequence comprising SEQ ID NO:8;-   (e) a variable heavy chain sequence comprising SEQ ID NO:9 and a    variable light chain sequence comprising SEQ ID NO:10;-   (f) a variable heavy chain sequence comprising SEQ ID NO:11 and a    variable light chain sequence comprising SEQ ID NO:12;-   (g) a variable heavy chain sequence comprising SEQ ID NO:13 and a    variable light chain sequence comprising SEQ ID NO:14;-   (h) a variable heavy chain sequence comprising SEQ ID NO:15 and a    variable light chain sequence comprising SEQ ID NO:16;-   (i) a variable heavy chain sequence comprising SEQ ID NO:17 and a    variable light chain sequence comprising SEQ ID NO:18;-   (j) a variable heavy chain sequence comprising SEQ ID NO:19 and a    variable light chain sequence comprising SEQ ID NO:20;-   (k) a variable heavy chain sequence comprising SEQ ID NO:21 and a    variable light chain sequence comprising SEQ ID NO:22;-   (l) a variable heavy chain sequence comprising SEQ ID NO:23 and a    variable light chain sequence comprising SEQ ID NO:24;-   (m) a variable heavy chain sequence comprising SEQ ID NO:25 and a    variable light chain sequence comprising SEQ ID NO:26;-   (n) a variable heavy chain sequence comprising SEQ ID NO:27 and a    variable light chain sequence comprising SEQ ID NO:28;-   (o) a variable heavy chain sequence comprising SEQ ID NO:29 and a    variable light chain sequence comprising SEQ ID NO:30;-   (p) a variable heavy chain sequence comprising SEQ ID NO:31 and a    variable light chain sequence comprising SEQ ID NO:32;-   (q) a variable heavy chain sequence comprising SEQ ID NO:33 and a    variable light chain sequence comprising SEQ ID NO:34;-   (r) a variable heavy chain sequence comprising SEQ ID NO:35 and a    variable light chain sequence comprising SEQ ID NO:36;-   (s) a variable heavy chain sequence comprising SEQ ID NO:37 and a    variable light chain sequence comprising SEQ ID NO:38;-   (t) a variable heavy chain sequence comprising SEQ ID NO:39 and a    variable light chain sequence comprising SEQ ID NO:40;-   (u) a variable heavy chain sequence comprising SEQ ID NO:41 and a    variable light chain sequence comprising SEQ ID NO:42;-   (v) a variable heavy chain sequence comprising SEQ ID NO:43 and a    variable light chain sequence comprising SEQ ID NO:44; and-   (w) a variable heavy chain sequence comprising SEQ ID NO:45 and a    variable light chain sequence comprising SEQ ID NO:46.

In some embodiments, the provided antibodies are fully humananti-CLDN18.2 antibodies, or antigen-binding fragment thereof,comprising a specific pair of variable heavy chain and variable lightchain sequences, selected from the following combinations:

-   (aa) a variable heavy chain sequence comprising SEQ ID NO: 185 and a    variable light chain sequence comprising SEQ ID NO: 186;-   (bb) a variable heavy chain sequence comprising SEQ ID NO: 187 and a    variable light chain sequence comprising SEQ ID NO: 188;-   (cc) a variable heavy chain sequence comprising SEQ ID NO: 189 and a    variable light chain sequence comprising SEQ ID NO: 190;-   (dd) a variable heavy chain sequence comprising SEQ ID NO: 191 and a    variable light chain sequence comprising SEQ ID NO: 192;-   (ee) a variable heavy chain sequence comprising SEQ ID NO: 193 and a    variable light chain sequence comprising SEQ ID NO: 194;-   (ff) a variable heavy chain sequence comprising SEQ ID NO: 195 and a    variable light chain sequence comprising SEQ ID NO: 196;-   (gg) a variable heavy chain sequence comprising SEQ ID NO: 197 and a    variable light chain sequence comprising SEQ ID NO: 198;-   (hh) a variable heavy chain sequence comprising SEQ ID NO: 199 and a    variable light chain sequence comprising SEQ ID NO: 200;-   (ii) a variable heavy chain sequence comprising SEQ ID NO: 201 and a    variable light chain sequence comprising SEQ ID NO: 202;-   (jj) a variable heavy chain sequence comprising SEQ ID NO: 203 and a    variable light chain sequence comprising SEQ ID NO: 204;-   (kk) a variable heavy chain sequence comprising SEQ ID NO: 205 and a    variable light chain sequence comprising SEQ ID NO: 206;-   (ll) a variable heavy chain sequence comprising SEQ ID NO: 207 and a    variable light chain sequence comprising SEQ ID NO: 208;-   (mm) a variable heavy chain sequence comprising SEQ ID NO: 209 and a    variable light chain sequence comprising SEQ ID NO: 210;-   (nn) a variable heavy chain sequence comprising SEQ ID NO: 211 and a    variable light chain sequence comprising SEQ ID NO: 212;-   (oo) a variable heavy chain sequence comprising SEQ ID NO: 215 and a    variable light chain sequence comprising SEQ ID NO: 216;-   (pp) a variable heavy chain sequence comprising SEQ ID NO: 217 and a    variable light chain sequence comprising SEQ ID NO: 218; and-   (qq) a variable heavy chain sequence comprising SEQ ID NO: 219 and a    variable light chain sequence comprising SEQ ID NO: 220.

The skilled person will further understand that the variable light andvariable heavy chains may be independently selected, or mixed andmatched, from the antibodies provided herein.

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a combination of CDR sequences derived from a variableheavy chain sequence selected from the group consisting of SEQ NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43 and 45 and a variable light chain sequence selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46,

In some embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a combination of CDR sequences derived from a variableheavy chain sequence selected from the group consisting of SEQ lD NOs:185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,215, 217 and 219 and a variable light chain sequence selected from thegroup consisting of SEQ ID NOs: 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 216, 218 and 220.

In some embodiments, the anti-CLDN18 antibodies and antibody fragmentsthereof comprise one or more heavy chain variable region CDRs disclosedin Table 1 and/or one or more light chain variable region CDRs disclosedin Table 3.

In some embodiments, the anti-CLDN18 antibodies and antibody fragmentsthereof comprise one or more heavy chain variable region CDRs disclosedin Table 2 and/or one or more light chain variable region CDRs disclosedin Table 4.

In some embodiments, the anti-CLDN18.2 antibody or antibody fragment isa recombinant antibody (e.g., a chimeric antibody, humanized antibody,or a bispecific antibody) comprising six (6) CDRs, all derived from theVH or VL domain of a single anti-CLDN18.2 antibody disclosed herein. Forexample, a binding agent may comprise all six of the CDR regions for theanti-CLDN18.2 antibody designated as Hu-1. In this instance, theantibody or binding agent would comprise the amino acid sequences of SEQID NOS: 221-223 and SEQ ID NOS: 224-226, representing the CDR1, CDR2 andCDR3 of the variable heavy and the CDR1, CDR2 and CDR3 sequences of thevariable light domains, respectively, of the Hu-1 antibody.

In some embodiments, the anti-CLDN18 antibodies or antibody fragmentsthereof bind to the first extracellular domain/loop of CLDN18.2 andexhibit one or more of the following properties binding preferentiallyto CLDN18 isoform 2 being naturally expressed by human tumor cells, notbinding to CLDN18 isoform 1 (CLDN18.1), efficiently internalized fromthe surface of CLDN18.2 positive cells after binding, capable ofdirecting the killing of CLDN18.2 positive cells by inducingantibody-dependent cellular cytotoxicity (ADCC) mediated lysis,complement-dependent cytotoxicity (CDC), or antibody-dependentphagocytosis (ADPC).

The present disclosure also provides methods for the treatment ofepithelial cell-derived primary and metastatic cancer comprisingadministering a composition or formulation that comprises ananti-CLDN18.2 antibody, a bispecific antibody comprising ananti-CLDN18.2-specific binding agent, or an antibody fragment thereof,and optionally another immune-based therapy, to a subject in needthereof. In some embodiments, the cancer is selected from solid tumor,gastric cancer, esophageal cancer, cancer of the gastroesophagealjunction, pancreatic cancer, cancer of the bile duct, lung cancer,ovarian cancer, colon cancer, hepatic cancer, head and neck cancer,gallbladder cancer. In some instances the other immune-based therapy isa checkpoint inhibitor.

The anti-CLDN18.2 antibodies of the disclosure can also be used fordeveloping antibody-based immunotherapeutics that rely onCLDN18.2-specific binding to direct patient effector cells (e.g., Tcells or NK cells) to tumors including bispecific T cell engagingantibodies, or bispecific molecules that redirect NK cells, or celltherapies, such as CAR-T therapy or for delivery to toxic payloads(e.g., a conjugated cytotoxic drug) to CLDN18.2 positive solid tumors.

The anti-CLDN18.2 antibodies of the disclosure can also be used fordiagnosis of gastric cancer, esophageal cancer, cancer of thegastroesophageal junction, pancreatic cancer, cancer of the bile duct,lung cancer, ovarian cancer, colon cancer, hepatic cancer, head and neckcancer, gallbladder cancer.

BRIEF DESCRIPTION OF THE OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,shown in the figures are embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements, examples, and instrumentalities shown.

FIG. 1 provides the amino acid sequences of the VH and VL domains of themurine anti-CLDN18.2 antibodies and their respective CDR sequences.Sequence identifiers are provided and the CDRs are underlined in thevariable domain sequences.

FIG. 2 provides the amino acid sequences of the VH and VL domains of thehuman anti-CLDN18.2 antibodies and their respective CDR sequences.Sequence identifiers are provided and the CDRs are underlined in thevariable domain sequences.

FIGS. 3A, 3B and 3C demonstrate the binding of mouse anti-Claudin18.2antibodies to CHO-Claudin18.2 cells. FIG. 3A shows the binding intensitycounts of murine anti-Claudin18.2 antibodies. FIG. 3B shows the bindingcurves of murine anti-Claudin18.2 antibodies Ms-1, Ms-2, and Ms-3 toCHO-Claudin18.2 cells. FIG. 3C shows the binding of Claudin18.2antibodies Ms-4 and Hu-3 to CHO-Cladin18.2 cells, but not toCHO-Cladin18.1.

FIGS. 4A and 4B are graphs showing the binding of human anti-Claudin18.2antibodies to CHO-Claudin18.2 and CHO-Claudin18.1 cells. FIG. 4A showsthe binding of human anti-Claudin18.2 antibodies. FIG. 4B shows thebinding curves of human anti-Claudin18.2 antibodies Hu-2, Hu-9, andHu-10 to CHO-Claudin18.2 cells.

FIG. 5 is a bar graph showing the comparison of the surface Claudin18.2expression level on CHO-Claudin18.2 and tumor cell lines PATU8988S andNUGC-4.

FIGS. 6A and 6B are bar graphs showing the binding of anti-Claudin18.2antibodies to tumor cell line PATU8988S. FIG. 6A shows the bindingintensity counts of murine anti-Claudin18.2 antibodies. FIG. 6B showsthe binding of human anti-Claudin18.2 antibodies.

FIG. 7 is a bar graph showing the binding of human anti-Claudin18.2antibodies to tumor cell line NUGC-4.

FIG. 8 is a bar graph showing the binding of human anti-Claudin18.2antibodies to tumor cell line KatoIII.

FIG. 9 demonstrates Hu-2-, Hu-7-, and Hu-9-mediated antibody-dependentcellular cytotoxicity (ADCC).

FIG. 10 demonstrates antibody-mediated endocytosis induced byanti-Claudin18.2 antibodies Hu-2, Hu-7, Hu-9, and Hu-11.

FIGS. 11A and 11B provide the amino acid sequences of four humanimmunoglobulin IgG1 CH domains (FIG. 11A) and a human immunoglobulinkappa light chain CL domain (FIG. 11B) suitable for use in combinationwith the disclosed anti-CLDN18.2 VH and VL domains and/or CDR sequences.Sequence identifiers are provided.

FIG. 12 provides the amino acid sequence of two representativefull-length anti-CLDN18.2 antibody) recombinant heavy chains comprisingthe amino acid sequence provided in SEQ ID NO: 336 or SEQ ID NO: 338,either of which can be paired with a recombinant kappa light chainhaving the amino acid sequence provided in SEQ ID NO: 337 to provide afull-length human IgG1 anti-CLDN18.2 antibody.

FIGS. 13A and 13B demonstrate Fc modified Claudin18.2 antibodiesNBL-014P and NBL-014G induce enhanced ADCC against CHO cellsoverexpressing human claudin18.2. using Promega surrogate effector cells(FIG. 13A) or NK92MI cells overexpressing CD16A (FIG. 13B).

FIGS. 14A and 14B show the Fc modified anti-Claudin18.2 antibodiesNBL-014P and NBL-014G induce enhanced ADCC against NUGC-4 cells (FIG.14A) and PATU8988S cells (FIG. 14B).

FIG. 15 shows the Fc modified anti-Claudin18.2 antibodies NBL-014P andNBL-014G induce enhanced CDC, compared to the CDC activity of PC1,against CHO cells overexpressing human claudin18.2.

FIG. 16 shows the Fc modified anti-Claudin18.2 antibodies NBL-014P andNBL-014G induce enhanced CDC activity, compared to the CDC activity ofPC1, against PATU8988S cells.

FIGS. 17A-17B show ADCP activity of anti-Claudin18.2 antibodies againstCHO cells overexpressing human claudin18.2 (FIG. 17A) and NUGC-4 cells(FIG. 17B).

FIGS. 18A and 18B demonstrate the anti-tumor efficacy of anti-Claudin18.2 antibodies NBL-014 and PC1 in a subcutaneous PATU8988Soverexpressing human Claudin 18.2 model based on determination ofchanges in tumor volume over time (FIG. 18A) and tumor weight on day 34(FIG. 18B).

FIG. 19 is a graph showing the anti-tumor efficacy of anti-Claudin 18.2antibodies NBL-014 and PC1 in a subcutaneous NUGC-4 model based onchanges in tumor volume over time.

FIGS. 20A and 20B demonstrate the anti-tumor efficacy of anti-Claudin18.2 antibody NBL-014P in a subcutaneous NUGC-4 model implanted in humanPBMC engrafted NOGdKO mice based on determination of changes in tumorvolume over time (FIG. 20A) and tumor weight on day 28 (FIG. 20B).

FIG. 21 provides the amino sequence of Human claudin 18 isoform 2(claudin 18.2) (SEQ ID NO: 329) and Human claudin 18 isoform 1 (claudin18.1) (SEQ ID NO: 330).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure generally relates to claudin 18.2(CLDN18.2)-specific antibodies and antibody fragments thereof. Theanti-CLDN18.2 antibodies or antibody fragments thereof, or bispecificmolecules, or fusion proteins comprising the CDRs or VH and VL sequencesof the disclosed anti-CLDN18.2 antibodies may be used for antibody-basedtherapies of diseases associated with dysregulation of CLDN18.2expression. More specifically, the anti-CLDN18.2 antibodies can be usedfor immunotherapy of epithelial cell-derived primary and metastaticcancer, including gastric, pancreatic and esophageal, lung, ovarian,colon, and hepatic cancers.

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

Throughout this disclosure the following abbreviations will be used.

-   ADC—Antibody drug conjugate.-   ADCC—Antibody-dependent cellular cytotoxicity.-   CDC—Complement-dependent cytotoxicity.-   CDR—Complementarity determining region in the immunoglobulin    variable regions.-   ECD—Extracellular domain.-   FR—Antibody framework region, the immunoglobulin variable regions    excluding the-   CDR regions.-   EC50—the concentration of antibody that gives half-maximal binding    or the efficient concentration of an antibody which produces 50% of    its maximum response.-   IC50—the concentration resulting in 50% inhibition.-   mAb or Mab or MAb—Monoclonal antibody.-   VH or VII—Immunoglobulin heavy chain variable region.-   VL or VL—Immunoglobulin light chain variable region.-   eADCC—Enhanced Antibody-dependent cellular cytotoxicity.-   ADCP—Antibody-dependent cellular phagocytosis.-   Fc—Fragment crystallizable region.-   FcRn—the neonatal Fc receptor.

As used herein the term “CLDN” means claudin and includes CLDN18.2 andCLDN 18.1. Preferably, a claudin is a human claudin.

As used herein, the term “claudin 18 isoform 2” (used interchangeablywith CLDN18.2) refers to a peptide comprising or consisting of the aminoacid sequence provided in Uni-Prot entry P56856 (CLD18 human) identifiedas P56856-2, isoform (splice variant 2) including post-translationallymodified variants and species homologs present on the surface of normalor transformed cancer cells or are expressed on cells transfected with aCLDN18.2 gene. Claudin 18.2 preferably has the amino acid sequenceaccording to SEQ ID NO: 329.

As used herein, the term “claudin 18 isoform 1” (used interchangeablywith CLDN18.1) refers to a peptide comprising or consisting of the aminoacid sequence provided in Uni-Prot entry P56856 (CLD18_human) identifiedas P56856-1 isoform (splice variant 1), including post-translationallymodified variants and species homologs of present on the surface ofnormal or transformed cancer cells or are expressed on cells transfectedwith a CLDN18.1 gene. Claudin 18.1 preferably has the amino acidsequence according to SEQ ID NO: 330.

The term “extracellular domain” or “extracellular portion” as usedherein refers to a part of a molecule such as a protein that is facingthe extracellular space of a cell and preferably is accessible from theoutside of said cell, e.g., by antigen-binding molecules such asantibodies located outside the cell. Preferably, the term refers to oneor more extracellular loops or domains or a fragment thereof.

As used herein, the term “CLDN18-associated disease or disorder”includes disease states and/or symptoms associated with a disease state,where altered levels or activity, or accessibility of CLDN18.2 arefound. Exemplary CLDN18.2-associated diseases or disorders include, butare not limited to, cancer diseases associated with epithelial tumorsexpressing CLDN18.2, such as pancreatic and gastroesophageal cancers.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies).

An exemplary antibody such as an IgG comprises two heavy chains and twolight chains. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region.Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged from aminoterminus to carboxy-terminus in the following order: FR1, CDR1, FR2,CDR2, FR3, CDR3, FR4.

The “class” or “isotype” of an antibody refers to the type of constantdomain or constant region possessed by its heavy chain. There are fivemajor classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several ofthese may be further divided into subclasses (isotypes), e.g., IgG1,IgG2, IgG3, IgG1 4, IgA1, and IgA2. The heavy chain constant domainsthat correspond to the different classes of immunoglobulins are calleda, 6, c, y, and respectively.

As used herein the term “IgG” refers to the isotype of humanimmunoglobulin comprising antibodies belonging to the four subclassesIgG1, IgG2, IgG3 and IgG1 4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35 (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies and is not to be construed as requiringproduction of the antibody by any method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including but not limited to thehybridoma method, recombinant DNA methods, phage-display methods, andmethods utilizing transgenic animals containing all or part of the humanimmunoglobulin loci, such methods and other exemplary methods for makingmonoclonal antibodies being described herein.

The term “chimeric” antibody refers to a recombinant antibody in which aportion of the heavy and/or light chain is derived from a particularsource or species, while the remainder of the heavy and/or light chainis derived from a different source or species.

A “human antibody” is an antibody that possesses an amino acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies known toone of skill in the art. This definition of a human antibodyspecifically excludes a humanized antibody comprising non-humanantigen-binding residues. Human antibodies can be produced using varioustechniques known in the art, including methods described in Cole et al,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boerner et al, J. Immunol, 147(I):86-95 (1991). See also van Dijk andvan de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodiescan be prepared by administering the antigen to a transgenic animal thathas been modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedHuMab mice (see, e.g., Nils Lonberg et al., 1994, Nature 368:856-859, WO98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO01/09187 regarding HuMab mice), xenomice (see, e.g., U.S. Pat. Nos.6,075,181 and 6,150,584 regarding XENOMOUSE™ technology) or Trianni mice(see, e.g., WO 2013/063391, WO 2017/035252 and WO 2017/136734 regardingTrianni mice).

The term “humanized antibody” refers to an antibody that has beenengineered to comprise one or more human framework regions in thevariable region together with non-human (e.g., mouse, rat, or hamster)complementarity-determining regions (CDRs) of the heavy and/or lightchain. In certain embodiments, a humanized antibody comprises sequencesthat are entirely human except for the CDR regions. Humanized antibodiesare typically less immunogenic to humans, relative to non-humanizedantibodies, and thus offer therapeutic benefits in certain situations.Those skilled in the art will be aware of humanized antibodies and willalso be aware of suitable techniques for their generation. See forexample, Hwang, W. Y. K., et al., Methods 36:35, 2005; Queen et al.,Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature,321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyenet al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad.Sci. USA, 86:3833-37, 1989; U.S. Pat. Nos. 5,225,539; 5,530,101;5,585,089; 5,693,761; 5,693,762; 6,180,370; and Selick et al., WO90/07861, each of which is incorporated herein by reference in itsentirety.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody comprising a heavy chain variable domain(VH) and a light chain variable domain (VL), where the VH-VL unit haspolyepitopic specificity (i.e., is capable of binding to two differentepitopes on one biological molecule or each epitope on a differentbiological molecule). Such multispecific antibodies include, but are notlimited to, full-length antibodies, antibodies having two or more VL andVH domains, bispecific diabodies and triabodies. “Polyepitopicspecificity” refers to the ability to specifically bind to two or moredifferent epitopes on the same or different target(s).

“Dual specificity” or “bispecificity” refers to the ability tospecifically bind to two different epitopes on the same or differenttarget(s). However, in contrast to bispecific antibodies, dual-specificantibodies have two antigen-binding arms that are identical in aminoacid sequence and each Fab arm is capable of recognizing two antigens.Dual-specificity allows the antibodies to interact with high affinitywith two different antigens as a single Fab or IgG molecule. Accordingto one embodiment, the multispecific antibody in an IgG1 form binds toeach epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1μM to 0.001 pM, 0.5 μM to 0.001 pM or 0.1 μM to 0.001 pM. “Monospecific”refers to the ability to bind only one epitope. Multi-specificantibodies can have structures similar to full immunoglobulin moleculesand include Fc regions, for example IgG Fc regions. Such structures caninclude, but are not limited to, IgG-Fv, IgG-(scFv)2, DVD-Ig,(scFv)2-(scFv)2-Fc and (scFv)2-Fc-(scFv)2. In case of IgG-(scFv)2, thescFv can be attached to either the N-terminal or the C-terminal end ofeither the heavy chain or the light chain.

As used herein, the term “bispecific antibodies” refers to monoclonal,often human or humanized, antibodies that have binding specificities forat least two different antigens. In the invention, one of the bindingspecificities can be directed towards CLDN18.2, the other can be for anyother antigen, e.g., for a cell-surface protein, receptor, receptorsubunit, tissue-specific antigen, virally derived protein, virallyencoded envelope protein, bacterially derived protein, or bacterialsurface protein, etc.

As used herein, the term “diabodies” refers to bivalent antibodiescomprising two polypeptide chains, in which each polypeptide chainincludes VH and VL domains joined by a linker that is too short (e.g., alinker composed of five amino acids) to allow for intramolecularassociation of VH and VL domains on the same peptide chain. Thisconfiguration forces each domain to pair with a complementary domain onanother polypeptide chain so as to form a homodimeric structure.Accordingly, the term “triabodies” refers to trivalent antibodiescomprising three peptide chains, each of which contains one VH domainand one VL domain joined by a linker that is exceedingly short (e.g., alinker composed of 1 -2 amino acids) to permit intramolecularassociation of VH and VL domains within the same peptide chain.

The term “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab)₂;diabodies; linear antibodies; single-chain antibody molecules (e.g.,scFv). Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, and a residual “Fc”fragment, a designation reflecting the ability to crystallize readily.The Fab fragment consists of an entire light (L) chain (VL) along withthe variable region domain of the heavy (H) chain (VII), and the firstconstant domain of one heavy chain (CH1). Pepsin treatment of anantibody yields a single large F(ab)₂ fragment which roughly correspondsto two disulfide linked Fab fragments having divalent antigen-bindingactivity and is still capable of cross-linking antigen. Fab fragmentsdiffer from F(ab)₂ fragments by having additional few residues at thecarboxy terminus of the CH1 domain including one or more cysteines fromthe antibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The terms “antigen-binding domain” or “antigen-binding portion” of anantibody (or more simply “binding domain” or “binding portion”) orsimilar terms refer to one or more fragments of an antibody that retainthe ability to specifically bind to an antigen complex. Examples ofbinding fragments encompassed within the term “antigen-binding portion”of an antibody include (i) Fab fragments, monovalent fragmentsconsisting of the VL, VH, CL and CH domains; (ii) F(ab′)2 fragments,bivalent fragments comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) Fd fragments consisting of the VH andCH domains; (iv) Fv fragments consisting of the VL and VH domains of asingle arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature341: 544-546), which consist of a VH domain; (vi) isolatedcomplementarity determining regions (CDR), and (vii) combinations of twoor more isolated CDRs which may optionally be joined by a syntheticlinker.

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Complementarity determining region” or “CDR” as the terms are usedherein refer to short polypeptide sequences within the variable regionof both heavy and light chain polypeptides that are primarilyresponsible for mediating specific antigen recognition. There are threeCDRs (termed CDR1, CDR2, and CDR3) within each VL and each VH.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated CDRs. Accordingly, the disclosure of each variable heavyregion is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2 and vhCDR3)and the disclosure of each variable light region is a disclosure of thevlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).

In certain embodiments, the CDRs of an antibody can be determinedaccording to the IMGT numbering system as described in Lefranc M-P,(1999) The Immunologist 7: 132-136 and Lefranc M-P et al, (1999) NucleicAcids Res 27: 209-212, each of which is herein incorporated by referencein its entirety. Unless stated otherwise herein, references to residuenumbers in the variable domain of antibodies mean residue numbering bythe IMGT numbering system.

In other embodiments, the CDRs of an antibody can be determinedaccording to MacCallum R M et al, (1996) J Mol Biol. 262: 732-745,herein incorporated by reference in its entirety. See also, e.g. MartinA. “Protein Sequence and Structure Analysis of Antibody VariableDomains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter31, pp. 422-439, Springer-Verlag, Berlin (2001), herein incorporated byreference in its entirety. In other embodiments, the CDRs of an antibodycan be determined according to the AbM numbering scheme, which refers toAbM hypervariable regions, which represent a compromise between theKabat CDRs and Chothia structural loops, and are used by OxfordMolecular's AbM antibody modeling software (Oxford Molecular Group,Inc.), herein incorporated by reference in its entirety.

“Framework” or “framework region” or “FR” refers to variable domainresidues other than the hypervariable region (HVR) residues. The FR of avariable domain generally consists of four FR domains: FR1, FR2, FR3,and FR4.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup Illas in Kabat et al., supra.

The “hinge region” is generally defined as stretching from 216-238 (EUnumbering) or 226-251 (Kabat numbering) of human IgG1 . The hinge can befurther divided into three distinct regions, the upper, middle (e.g.,core), and lower hinge.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region and interacts with endogenous receptors present on thesurface of cells of the immune system and some proteins of thecomplement system. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, thenumbering of amino acid residues in the Fc region or constant region isaccording to the EU numbering system, also called the EU index, asdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991).

The term “isolated antibody” when used to describe the variousantibodies disclosed herein, means an antibody that has been identifiedand separated and/or recovered from a cell or cell culture from which itwas expressed. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide, and can include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In some embodiments, anantibody is purified to greater than 95% or 99% purity as determined by,for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing(IEF), capillary electrophoresis) or chromatographic (e.g., ion exchangeor reverse phase HPLC) approaches. For a review of methods forassessment of antibody purity, see, for example, Flatman et al., J.Chromatogr. B 848:79-87 (2007). In preferred embodiments, the antibodywill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes antibodies in situ withinrecombinant cells because at least one component of the polypeptidenatural environment will not be present.

An “epitope” is a term of art that indicates the site or sites ofinteraction between an antibody and its antigen(s). As described by(Janeway, C, Jr., P. Travers, et al. (2001). Immunobiology: the immunesystem in health and disease. Part II, Section 3-8. New York, GarlandPublishing, Inc.): “An antibody generally recognizes only a small regionon the surface of a large molecule such as a protein . . . [Certainepitopes] are likely to be composed of amino acids from different partsof the [antigen] polypeptide chain that have been brought together byprotein folding. Antigenic determinants of this kind are known asconformational or discontinuous epitopes because the structurerecognized is composed of segments of the protein that are discontinuousin the amino acid sequence of the antigen but are brought together inthe three-dimensional structure. In contrast, an epitope composed of asingle segment of polypeptide chain is termed a continuous or linearepitope” (Janeway, C., Jr., P. Travers, et al. (2001). Immunobiology:the immune system in health and disease. Part II Section 3-8. New York,Garland Publishing, Inc.).

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that contacts an overlapping set of amino acidresidues of the antigen as compared to the reference antibody or blocksbinding of the reference antibody to its antigen in a competition assayby 50% or more. The amino acid residues of an antibody that contact anantigen can be determined, for example, by determining the crystalstructure of the antibody in complex with the antigen or by performinghydrogen/deuterium exchange. In some embodiments, residues of anantibody that are within 5 A the antigen are considered to contact theantigen. In some embodiments, an antibody that binds to the same epitopeas a reference antibody blocks binding of the reference antibody to itsantigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

With regard to the binding of an antibody to a target molecule, the term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetmeans binding that is measurably different from a non-specificinteraction. For example, as used herein the terms “specific binding,”“specifically binds,” and “selectively binds,” refer to antibody bindingto an epitope of human CLDN18.2 where the binding is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tothe binding of a control molecule. For example, specific binding can bedetermined by competition with a control molecule that is similar to thetarget, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by the excess unlabeled target. Theterm “specific binding” or “specifically binds to” or is “specific for”a particular polypeptide or an epitope on a particular polypeptidetarget as used herein can be exhibited, for example, by a moleculehaving a KD for the target of 10-4 M or lower, alternatively 10-5 M orlower, alternatively 10-6 M or lower, alternatively 10-7 M or lower,alternatively 10-8 M or lower, alternatively 10-9 M or lower,alternatively 10-10 M or lower, alternatively 10-11 M or lower,alternatively 10-12 M or lower or a KD in the range of 10-4 M to 10-6 Mor 10-6 M to 10-10 M or 10-7 M to 10-9 M. As will be appreciated by theskilled artisan, affinity and KD values are inversely related. A highaffinity for an antigen is measured by a low KD value. In oneembodiment, the term “specific binding” refers to binding where amolecule binds to a particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

The term “affinity,” as used herein, means the strength of the bindingof an antibody to an epitope. The affinity of an antibody is given bythe dissociation constant Kd, defined as [Ab]×[Ag]/[Ab-Ag], where[Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab]is the molar concentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant Ka isdefined by 1/Kd. Methods for determining the affinity of mAbs can befound in Harlow, et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan etal., eds., Current Protocols in immunology, Greene Publishing Assoc. andWiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Envy mol.92:589-601 (1983), which references are entirely incorporated herein byreference. One standard method well known in the art for determining theaffinity of mAbs is the use of surface plasmon resonance (SPR) screening(such as by analysis with a BIAcore™ SPR analytical device).

“EC50” with respect to an agent and a particular activity (e.g. bindingto a cell, inhibition of enzymatic activity, activation or inhibition ofan immune cell), refers to the efficient concentration of the agentwhich produces 50% of its maximum response or effect with respect tosuch activity. “EC100” with respect to an agent and a particularactivity refers to the efficient concentration of the agent whichproduces its substantially maximum response with respect to suchactivity.

The term “effector functions” refer to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include C1qbinding and complement-dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down-regulation of cell surface receptors (e.g., B cellreceptor); and B cell activation.

As used herein the term “Fc y receptor” refers to a family of endogenousreceptors located in the membranes of immune cells including Blymphocytes, natural killer cells, macrophages, neutrophils, and mastcells which binds the Fc region of antibodies and is involved in antigenrecognition.

As used herein, the term “specifically binds” when referring to abinding pair (e.g. antibody/cell surface molecule), indicates a bindingreaction which is determinative of the presence of the protein, e.g.,CLDN18.2, in a heterogeneous population of proteins and/or otherbiologics. Thus, under designated conditions, a specified antibody bindsto a particular cell surface marker and does not bind in a significantamount to other proteins present on a cell surface or in a sample.

As used herein the term “specifically binds CLDN18.2” refers to theability of an antibody, or antigen-binding fragment to recognize andbind endogenous claudin 18 isoform 2 as it occurs on the surface ofnormal or malignant cells, but not to claudin 18 isoform 1 (CLDN18.1),or to any other lineage-specific cell surface marker.

As used herein the term “endocytosis” refers to the process whereeukaryotic cells internalize segments of the plasma membrane,cell-surface receptors, and components from the extracellular fluid.Endocytosis mechanisms include receptor-mediated endocytosis. The term“receptor-mediated endocytosis” refers to a biological mechanism bywhich a ligand, upon binding to its target, triggers membraneinvagination and pinching, gets internalized and delivered into thecytosol or transferred to appropriate intracellular compartments.

As used herein the terms “antibody-based immunotherapy” and“immunotherapies” are used to broadly refer to any form of therapy thatrelies on the targeting specificity of an anti-CLDN18.2 antibody,bispecific molecule, antigen-binding domain, or fusion proteincomprising a CLDN18.2 antibody or antibody fragments or CDRs thereof, tomediate a direct or indirect effect on a cell characterized by aberrantexpression of CLDN18.2. The terms are meant to encompass methods oftreatment using naked antibodies, bispecific antibodies (including Tcell engaging, NK cell engaging and other immune cell/effector cellengaging formats) antibody drug conjugates, cellular therapies using Tcells (CAR-T) or NK cells (CAR-NK) engineered to comprise aCLDN18.2-specific chimeric antigen receptor and oncolytic virusescomprising a CLDN18.2 specific binding agent, and gene therapies bydelivering the antigen binding sequences of the anti-CLDN18.2 antibodiesand express the corresponding antibody fragments in vivo.

It is noted here that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

Claudin 18 (CLDN18)

The claudin family of proteins were first cloned and named in 1998 ascrucial structural and functional components of tight junctions. As afamily, claudins are a multigene family of tetra-transmembrane proteinsinvolved in the barrier functions of epithelial and endothelial cellsand the maintenance of the cytoskeleton (Furuse et al., J. Cell. Biol.141(7):1539-50, 1998). CLDN18 has different variants or conformations,including CLDN18 isoform 1 (CLDN18.1) and CLDN18 isoform 2 (CLDN18.2).

The first extracellular domain (ECD) of a claudin protein typicallyconsists of about 50 amino acids, while the second one is smaller havingabout 22 amino acids (Hashimoto, et al. Drug Discovery Today21(10):1711-1718, 2016). The N-terminal end is usually very short (e.g.about four to ten amino acids) while the C-terminal end ranges from 21to about 63 amino acids and is required for localization of the proteinsin tight junctions.

The observation that tight junction permeability is often higher intumor tissues than in normal tissues, has led to speculation thatclaudin proteins on tumor cells may be more accessible than in normaltissues with intact tight junctions. This observation also makes claudinproteins attractive targets for therapeutic cancer interventions. Inaddition, published expression profiling results suggest that cancertherapies targeting CLDN18.2 will have favorable systemic toxicityprofiles because normal turnover and homeostasis processes replenishgastrointestinal epithelial cells every two to seven days (Sahin et al).Transient gastrointestinal toxicity of limited duration is a common andmanageable adverse event for cancer immunotherapeutics.

CLDN18.2 comprises four membrane spanning domains with two smallextracellular loops (loop 1 embraced by hydrophobic region 1 andhydrophobic region 2; loop 2 embraced by hydrophobic regions 3 and 4).CLDN18.2 is a transmembrane protein, therefore epitopes present within,or formed by, its extracellular loops represent desirable targets forantibody-based cancer immunotherapy. However, given that CLDN18.1 isexpressed by alveolar epithelial cells in normal lung tissue, which is atissue that is highly relevant to toxicity, exclusive splice variantspecificity was a recognized prerequisite for the use ofCLDN18.2-specific antibodies for antibody-based cancer immunotherapy.Sahin et al were the first to report proof-of-concept results validatingCLDN18.2 as a druggable target for cancer immunotherapies based on theisolation of antibodies (polyclonal and monoclonal) that exclusivelybind to CLDN18.2 and not to CLDN18.1 (Sahin et al., Clin. Cancer Res. 14(23) 7624-7634, 2008).

CLDN18.2 is expressed in a number of primary tumors and theirmetastasis, including gastric cancer, esophageal cancer, pancreaticcancer, lung cancer such as non-small cell lung cancer, ovarian cancer,colon cancer, hepatic cancer, head-neck cancer, and cancers of the gallbladder. Dysregulated expression of claudins are detected in manycancers and may contribute to tumorigenesis and cancer invasiveness(Singh et al., J Oncology 2010; 2010: 541957). The expression ofCLDN18.2 is notably elevated in pancreatic ductal adenocarcinomas (PDAC)(Tanaka et al., J Histochem Cytochem. 2011; 59:942-952), esophagealtumors, non-small cell lung cancers (NSCLC), ovarian cancers (Sahin etal., Hu Cancer Biol. 2008; 14:7624-7634), and bile duct adenocarcinomas(Keira et al., Virchows Arch. 2015; 466:265-277).

Despite the fact that gastric cancer contributes to significantcancer-related morbidity and mortality, the treatment options forgastric cancer are limited. Claudins are present in normal tissues,benign neoplasms, hyperplastic conditions and cancers (Ding et al.,Cancer Manag. Res. 5:367-375 (2013)). The expression pattern of claudinsis highly tissue-specific, and most tissues express multiple claudins.Claudin proteins can interact with claudins from adjacent cells in ahomotypic or heterotypic fashion to form tight junctions (Ding et al.).Alterations in claudin expression and signaling pathways are known to beassociated with cancer development and an association between thefunction of impaired tight junctions and tumor progression has beenwidely reported.

Anti-CLDN18.2 Antibodies

Anti-CLDN18 antibodies, in particular human and murine monoclonalanti-CLDN18.2 antibodies, that specifically bind to the firstextracellular domain/loop of CLDN18.2 are provided. Binding of theCLDN18.2 monoclonal antibody or antigen binding fragment to CLDN18.2 canmediate complement-dependent cytotoxicity (CDC), antibody-dependentphagocytosis (ADPC), and/or antibody-dependent cellular cytotoxicity(ADCC) or other effects that result in the death of the targeted cancercell. Alternatively, the antibody or antigen binding fragment thereofcan, for example, serve to deliver a conjugated cytotoxic drug, and/orcan form a bispecific antibody with another monoclonal antibody tomediate the death of the targeted cancer cell.

In an embodiment, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a VH having a set of CDRs (HCDR1, HCDR2, and HCDR3)disclosed in Table 1 or Table 2. For example, the anti-CLDN18 antibodiesor antibody fragments thereof may comprise a set of CDRs correspondingto those CDRs in one or more of the anti-CLDN18.2 antibody heavy chainsdescribed in Table 2 (e.g., the CDRs of the Hu-1 antibody).

In another embodiment, the anti-CLDN18.2 antibodies comprise a VL havinga set of CDRs (LCDR1, LCDR2, and LCDR3) as disclosed in Table 3 or Table4. For example, the anti-CLDN18 antibodies or antibody fragments thereofmay comprise a set of CDRs corresponding to those CDRs in one or more ofthe anti-CLDN18.2 antibody light chains described in Table 4 (e.g., theCDRs of the Hu-1 antibody).

In an alternative embodiment, the anti-CLDN18 antibodies or antibodyfragments thereof comprise a VH having a set of CDRs (HCDR1, HCDR2, andHCDR3) as disclosed in Table 1, and a VL having a set of CDRs (LCDR1,LCDR2, and LCDR3) as disclosed in Table 3.

In an alternative embodiment, the anti-CLDN18 antibodies or antibodyfragments thereof comprise a VH having a set of CDRs (HCDR1, HCDR2, andHCDR3) as disclosed in Table 2, and a VL having a set of CDRs (LCDR1,LCDR2, and LCDR3) as disclosed in Table 4.

In a particular embodiment the anti-CLDN18.2 antibody may comprise a setof six (6) CDRs derived from a single murine antibody (VH CDRs in Table1 and VL CDRs in Table 3) or fully human antibody (VH CDRs in Table 2and VL CDRs in Table 4). For example, the antibody may comprise a set ofsix CDR regions derived from the fully human anti-CLDN18.2 antibody“Hu-1.” In this instance, the binding agent would comprise the aminoacid sequences of SEQ ID NOS: 221-223 and SEQ ID NOS: 224-226,representing the CDR1, CDR2 and CDR3 of the variable heavy and the CDR1,CDR2 and CDR3 sequences of the variable light domains, respectively, ofthe Hu-1 antibody.

TABLE 1 CDR sequences of Murine Variable Heavy Chain Domainsanti-CLDN18.2 mAb CDR1 CDR2 CDR3 Ms1 SEQ ID NO: 47 SEQ ID NO: 48 SEQ IDNO: 49 Ms2 SEQ ID NO: 53 SEQ ID NO: 54 SEQ ID NO: 55 Ms3 SEQ ID NO: 59SEQ ID NO: 60 SEQ ID NO: 61 Ms4 SEQ ID NO: 65 SEQ ID NO: 66 SEQ ID NO:67 Ms5 SEQ ID NO: 71 SEQ ID NO: 72 SEQ ID NO: 73 Ms6 SEQ ID NO: 77 SEQID NO: 78 SEQ ID NO: 79 Ms7 SEQ ID NO: 83 SEQ ID NO: 84 SEQ ID NO: 85Ms8 SEQ ID NO: 89 SEQ ID NO: 90 SEQ ID NO: 91 Ms9 SEQ ID NO: 95 SEQ IDNO: 96 SEQ ID NO: 97 Ms10 SEQ ID NO: 101 SEQ ID NO: 102 SEQ ID NO: 103Ms11 SEQ ID NO: 107 SEQ ID NO: 108 SEQ ID NO: 109 Ms12 SEQ ID NO: 113SEQ ID NO: 114 SEQ ID NO: 115 Ms13 SEQ ID NO: 119 SEQ ID NO: 120 SEQ IDNO: 121 Ms14 SEQ ID NO: 125 SEQ ID NO: 126 SEQ ID NO: 127 Ms15 SEQ IDNO: 131 SEQ ID NO: 132 SEQ ID NO: 133 Ms16 SEQ ID NO: 137 SEQ ID NO: 138SEQ ID NO: 139 Ms17 SEQ ID NO: 143 SEQ ID NO: 144 SEQ ID NO: 145 Ms18SEQ ID NO: 149 SEQ ID NO: 150 SEQ ID NO: 151 Ms19 SEQ ID NO: 155 SEQ IDNO: 156 SEQ ID NO: 157 Ms20 SEQ ID NO: 161 SEQ ID NO: 162 SEQ ID NO: 163Ms21 SEQ ID NO: 167 SEQ ID NO: 168 SEQ ID NO: 169 Ms22 SEQ ID NO: 173SEQ ID NO: 174 SEQ ID NO: 175 Ms23 SEQ ID NO: 179 SEQ ID NO: 180 SEQ IDNO: 181

TABLE 2 CDR sequences of Human Variable Heavy Chain Domainsanti-CLDN18.2 mAb CDR1 CDR2 CDR3 Hu-1 SEQ ID NO: 221 SEQ ID NO: 222 SEQID NO: 223 Hu-2 SEQ ID NO: 227 SEQ ID NO: 228 SEQ ID NO: 229 Hu-3 SEQ IDNO: 233 SEQ ID NO: 234 SEQ ID NO: 235 Hu-4 SEQ ID NO: 239 SEQ ID NO: 240SEQ ID NO: 241 Hu-5 SEQ ID NO: 245 SEQ ID NO: 246 SEQ ID NO: 247 Hu-6SEQ ID NO: 251 SEQ ID NO: 252 SEQ ID NO: 253 Hu-7 SEQ ID NO: 257 SEQ IDNO: 258 SEQ ID NO: 259 Hu-8 SEQ ID NO: 263 SEQ ID NO: 264 SEQ ID NO: 265Hu-9 SEQ ID NO: 269 SEQ ID NO: 270 SEQ ID NO: 271 Hu-10 SEQ ID NO: 275SEQ ID NO: 276 SEQ ID NO: 277 Hu-11 SEQ ID NO: 281 SEQ ID NO: 282 SEQ IDNO: 283 Hu-12 SEQ ID NO: 287 SEQ ID NO: 288 SEQ ID NO: 289 Hu-13 SEQ IDNO: 293 SEQ ID NO: 294 SEQ ID NO: 295 Hu-14 SEQ ID NO: 299 SEQ ID NO:300 SEQ ID NO: 301 Hu-16 SEQ ID NO: 311 SEQ ID NO: 312 SEQ ID NO: 313Hu-17 SEQ ID NO: 317 SEQ ID NO: 318 SEQ ID NO: 319 Hu-18 SEQ ID NO: 323SEQ ID NO: 324 SEQ ID NO: 325

TABLE 3 CDR sequences of Murine Variable Light Chain Domainsanti-CLDN18.2 mAb CDR1 CDR2 CDR3 Ms1 SEQ ID NO: 50 SEQ ID NO: 51 SEQ IDNO: 52 Ms2 SEQ ID NO: 56 SEQ ID NO: 57 SEQ ID NO: 58 Ms3 SEQ ID NO: 62SEQ ID NO: 63 SEQ ID NO: 64 Ms4 SEQ ID NO: 68 SEQ ID NO: 69 SEQ ID NO:70 Ms5 SEQ ID NO: 74 SEQ ID NO: 75 SEQ ID NO: 76 Ms6 SEQ ID NO: 80 SEQID NO: 81 SEQ ID NO: 82 Ms7 SEQ ID NO: 86 SEQ ID NO: 87 SEQ ID NO: 88Ms8 SEQ ID NO: 92 SEQ ID NO: 93 SEQ ID NO: 94 Ms9 SEQ ID NO: 98 SEQ IDNO: 99 SEQ ID NO: 101 Ms10 SEQ ID NO: 104 SEQ ID NO: 105 SEQ ID NO: 106Ms11 SEQ ID NO: 110 SEQ ID NO: 111 SEQ ID NO: 112 Ms12 SEQ ID NO: 116SEQ ID NO: 117 SEQ ID NO: 118 Ms13 SEQ ID NO: 122 SEQ ID NO: 123 SEQ IDNO: 124 Ms14 SEQ ID NO: 128 SEQ ID NO: 129 SEQ ID NO: 130 Ms15 SEQ IDNO: 134 SEQ ID NO: 135 SEQ ID NO: 136 Ms16 SEQ ID NO: 140 SEQ ID NO: 141SEQ ID NO: 142 Ms17 SEQ ID NO: 146 SEQ ID NO: 147 SEQ ID NO: 148 Ms18SEQ ID NO: 152 SEQ ID NO: 153 SEQ ID NO: 154 Ms19 SEQ ID NO: 158 SEQ IDNO: 159 SEQ ID NO: 160 Ms20 SEQ ID NO: 164 SEQ ID NO: 165 SEQ ID NO: 166Ms21 SEQ ID NO: 170 SEQ ID NO: 171 SEQ ID NO: 172 Ms22 SEQ ID NO: 176SEQ ID NO: 177 SEQ ID NO: 178 Ms23 SEQ ID NO: 182 SEQ ID NO: 183 SEQ IDNO: 184

TABLE 4 CDR sequences of Human Variable Light Chain Domainsanti-CLDN18.2 mAb CDR1 CDR2 CDR3 Hu-1 SEQ ID NO: 224 SEQ ID NO: 225 SEQID NO: 226 Hu-2 SEQ ID NO: 230 SEQ ID NO: 231 SEQ ID NO: 232 Hu-3 SEQ IDNO: 236 SEQ ID NO: 237 SEQ ID NO: 238 Hu-4 SEQ ID NO: 242 SEQ ID NO: 243SEQ ID NO: 244 Hu-5 SEQ ID NO: 248 SEQ ID NO: 249 SEQ ID NO: 250 Hu-6SEQ ID NO: 254 SEQ ID NO: 255 SEQ ID NO: 256 Hu-7 SEQ ID NO: 260 SEQ IDNO: 261 SEQ ID NO: 262 Hu-8 SEQ ID NO: 266 SEQ ID NO: 267 SEQ ID NO: 268Hu-9 SEQ ID NO: 272 SEQ ID NO: 273 SEQ ID NO: 274 Hu-10 SEQ ID NO: 278SEQ ID NO: 279 SEQ ID NO: 280 Hu-11 SEQ ID NO: 284 SEQ ID NO: 285 SEQ IDNO: 286 Hu-12 SEQ ID NO: 290 SEQ ID NO: 291 SEQ ID NO: 292 Hu-13 SEQ IDNO: 296 SEQ ID NO: 297 SEQ ID NO: 298 Hu-14 SEQ ID NO: 302 SEQ ID NO:303 SEQ ID NO: 304 Hu-16 SEQ ID NO: 314 SEQ ID NO: 315 SEQ ID NO: 316Hu-17 SEQ ID NO: 320 SEQ ID NO: 321 SEQ ID NO: 322 Hu-18 SEQ ID NO: 326SEQ ID NO: 327 SEQ ID NO: 328

In an embodiment, the antibody may be a monoclonal, chimeric, humanizedor human antibody, or antigen-binding portions thereof, or bispecific ormultispecific binding agent that specifically binds to human CLDN18isoform 2.

In another embodiment, the anti-CLDN18.2 antibodies or antibody fragmentcomprise defined sets/combinations of complementarity determining region(CDR) sequences derived from the disclosed VH and/or VL domains of theanti-CLDN18.2 antibodies.

In an embodiment, the anti-CLDN8.2 antibody or antibody fragment thereofexhibits one or more of the following properties: binding preferentiallyto CLDN18 isoform 2 expressed by human tumor cells, not binding toCLDN18 isoform 1 (CLDN18.1), efficiently internalized from the surfaceof CLDN18.2 positive cells after binding, and capable of directing thekilling of CLDN18.2 positive cells by inducing antibody-dependentcellular cytotoxicity (ADCC) mediated lysis.

In an embodiment, anti-CLDN18.2 specific antibodies or binding fragmentsthereof comprise a VH comprising a set of complementarity-determiningregions CDR1, CDR2, and CDR3 selected from the group consisting of:

-   (i) CDR1: SEQ ID NO: 47, CDR2: SEQ ID NO: 48, CDR3: SEQ ID NO: 49;-   (ii) CDR1: SEQ ID NO: 53, CDR2: SEQ ID NO: 54, CDR3: SEQ ID NO: 55;-   (iii) CDR1: SEQ ID NO: 59, CDR2: SEQ ID NO: 60, CDR3: SEQ ID NO: 61;-   (iv) CDR1: SEQ ID NO: 65, CDR2: SEQ ID NO: 66, CDR3: SEQ ID NO: 67;-   (v) CDR1: SEQ ID NO: 71, CDR2: SEQ ID NO: 72, CDR3: SEQ ID NO: 73;-   (vi) CDR1: SEQ ID NO: 77, CDR2: SEQ ID NO: 78, CDR3: SEQ ID NO: 79;-   (vii) CDR1: SEQ ID NO: 83, CDR2: SEQ ID NO: 84, CDR3: SEQ ID NO: 85;-   (viii) CDR1: SEQ ID NO: 89, CDR2: SEQ ID NO: 90, CDR3: SEQ ID NO:    91;-   (ix) CDR1: SEQ ID NO: 95, CDR2: SEQ ID NO: 96, CDR3: SEQ ID NO: 97;-   (x) CDR1: SEQ ID NO: 101, CDR2: SEQ ID NO: 102, CDR3: SEQ ID NO:    103;-   (xi) CDR1: SEQ ID NO: 107, CDR2: SEQ ID NO: 108, CDR3: SEQ ID NO:    109;-   (xii) CDR1: SEQ ID NO: 113, CDR2: SEQ ID NO: 114, CDR3: SEQ ID NO:    115;-   (xiii) CDR1: SEQ ID NO: 119, CDR2: SEQ ID NO: 120, CDR3: SEQ ID NO:    121;-   (xiv) CDR1: SEQ ID NO: 125, CDR2: SEQ ID NO: 126, CDR3: SEQ ID NO:    127;-   (xv) CDR1: SEQ ID NO: 131, CDR2: SEQ ID NO: 132, CDR3: SEQ ID NO:    133;-   (xvi) CDR1: SEQ ID NO: 137, CDR2: SEQ ID NO: 138, CDR3: SEQ ID NO:    139;-   (xvii) CDR1: SEQ ID NO: 143, CDR2: SEQ ID NO: 144, CDR3: SEQ ID NO:    145;-   (xviii) CDR1: SEQ ID NO: 149, CDR2: SEQ ID NO: 150, CDR3: SEQ ID NO:    151;-   (xix) CDR1: SEQ ID NO: 155, CDR2: SEQ ID NO: 156, CDR3: SEQ ID NO:    157;-   (xx) CDR1: SEQ ID NO: 161, CDR2: SEQ ID NO: 162, CDR3: SEQ ID NO:    163;-   (xxi) CDR1: SEQ ID NO: 167, CDR2: SEQ ID NO: 168, CDR3: SEQ ID NO:    169;-   (xxii) CDR1: SEQ ID NO: 173, CDR2: SEQ ID NO: 174, CDR3: SEQ ID NO:    175; and-   (xxiii) CDR1: SEQ ID NO: 179, CDR2: SEQ ID NO: 180, CDR3: SEQ ID NO:    181.

In an embodiment, anti-CLDN18.2 specific antibodies or binding fragmentsthereof comprise a VL comprising a set of complementarity-determiningregions CDR1, CDR2, and CDR3 selected from the group consisting of:

-   (i) CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, CDR3: SEQ ID NO: 52;-   (ii) CDR1: SEQ ID NO: 56, CDR2: SEQ ID NO: 57, CDR3: SEQ ID NO: 58;-   (iii) CDR1: SEQ ID NO: 62, CDR2: SEQ ID NO: 63, CDR3: SEQ ID NO: 64;-   (iv) CDR1: SEQ ID NO: 68, CDR2: SEQ ID NO: 69, CDR3: SEQ ID NO: 70;-   (v) CDR1: SEQ ID NO: 74, CDR2: SEQ ID NO: 75, CDR3: SEQ ID NO: 76;-   (vi) CDR1: SEQ ID NO: 80, CDR2: SEQ ID NO: 81, CDR3: SEQ ID NO: 82;-   (vii) CDR1: SEQ ID NO: 86, CDR2: SEQ ID NO: 87, CDR3: SEQ ID NO: 88;-   (viii) CDR1: SEQ ID NO: 92, CDR2: SEQ ID NO: 93, CDR3: SEQ ID NO:    94;-   (ix) CDR1: SEQ ID NO: 98, CDR2: SEQ ID NO: 99, CDR3: SEQ ID NO: 100;-   (x) CDR1: SEQ ID NO: 104, CDR2: SEQ ID NO: 105, CDR3: SEQ ID NO:    106;-   (xi) CDR1: SEQ ID NO: 110, CDR2: SEQ ID NO: 111, CDR3: SEQ ID NO:    112;-   (xii) CDR1: SEQ ID NO: 116, CDR2: SEQ ID NO: 117, CDR3: SEQ ID NO:    118;-   (xiii) CDR1: SEQ ID NO: 122, CDR2: SEQ ID NO: 123, CDR3: SEQ ID NO:    124;-   (xiv) CDR1: SEQ ID NO: 128, CDR2: SEQ ID NO: 129, CDR3: SEQ ID NO:    130;-   (xv) CDR1: SEQ ID NO: 134, CDR2: SEQ ID NO: 135, CDR3: SEQ ID NO:    136;-   (xvi) CDR1: SEQ ID NO: 140, CDR2: SEQ ID NO: 141, CDR3: SEQ ID NO:    142;-   (xvii) CDR1: SEQ ID NO: 146, CDR2: SEQ ID NO: 147, CDR3: SEQ ID NO:    148;-   (xviii) CDR1: SEQ ID NO: 152, CDR2: SEQ ID NO: 153, CDR3: SEQ ID NO:    154;-   (xix) CDR1: SEQ ID NO: 158, CDR2: SEQ ID NO: 159, CDR3: SEQ ID NO:    160;-   (xx) CDR1: SEQ ID NO: 164, CDR2: SEQ ID NO: 165, CDR3: SEQ ID NO:    166;-   (xxi) CDR1: SEQ ID NO: 170, CDR2: SEQ ID NO: 171, CDR3: SEQ ID NO:    172;-   (xxii) CDR1: SEQ ID NO: 176, CDR2: SEQ ID NO: 177, CDR3: SEQ ID NO:    178; and-   (xxiii) CDR1: SEQ ID NO: 182, CDR2: SEQ ID NO: 183, CDR3: SEQ ID NO:    184.

In an embodiment, antibodies having the ability of binding to CLDN18.2comprise a combination of VH and VL each comprising a set ofcomplementarity-determining regions CDR1, CDR2 and CDR3 selected fromthe group consisting of:

-   (i) VH: CDR1: SEQ ID NO: 47, CDR2: SEQ ID NO: 48, CDR3: SEQ ID NO:    49, VL: CDR1: SEQ ID NO: 50, CDR2: SEQ ID NO: 51, CDR3: SEQ ID NO:    52;-   (ii) VH: CDR1: SEQ ID NO: 53, CDR2: SEQ ID NO: 54, CDR3: SEQ ID NO:    55, VL: CDR1: SEQ ID NO: 56, CDR2: SEQ ID NO: 57, CDR3: SEQ ID NO:    58;-   (iii) VH: CDR1: SEQ ID NO: 59, CDR2: SEQ ID NO: 60, CDR3: SEQ ID NO:    61, VL: CDR1: SEQ ID NO: 62, CDR2: SEQ ID NO: 63, CDR3: SEQ ID NO:    64;-   (iv) VH: CDR1: SEQ ID NO: 65, CDR2: SEQ ID NO: 66, CDR3: SEQ ID NO:    67, VL: CDR1: SEQ ID NO: 68, CDR2: SEQ ID NO: 69, CDR3: SEQ ID NO:    70;-   (v) VH: CDR1: SEQ ID NO: 71, CDR2: SEQ ID NO: 72, CDR3: SEQ ID NO:    73, VL: CDR1: SEQ ID NO: 74, CDR2: SEQ ID NO: 75, CDR3: SEQ ID NO:    76;-   (vi) VH: CDR1: SEQ ID NO: 77, CDR2: SEQ ID NO: 78, CDR3: SEQ ID NO:    79, VL: CDR1: SEQ ID NO: 80, CDR2: SEQ ID NO: 81, CDR3: SEQ ID NO:    82;-   (vii) VH: CDR1: SEQ ID NO: 83, CDR2: SEQ ID NO: 84, CDR3: SEQ ID NO:    85, VL: CDR1: SEQ ID NO: 86, CDR2: SEQ ID NO: 87, CDR3: SEQ ID NO:    88;-   (viii) VH: CDR1: SEQ ID NO: 89, CDR2: SEQ ID NO: 90, CDR3: SEQ ID    NO: 91, VL: CDR1: SEQ ID NO: 92, CDR2: SEQ ID NO: 93, CDR3: SEQ ID    NO: 94;-   (ix) VH: CDR1: SEQ ID NO: 95, CDR2: SEQ ID NO: 96, CDR3: SEQ ID NO:    97, VL: CDR1: SEQ ID NO: 98, CDR2: SEQ ID NO: 99, CDR3: SEQ ID NO:    100;-   (x) VH: CDR1: SEQ ID NO: 101, CDR2: SEQ ID NO: 102, CDR3: SEQ ID NO:    103, VL: CDR1: SEQ ID NO: 104, CDR2: SEQ ID NO: 105, CDR3: SEQ ID    NO: 106;-   (xi) VH: CDR1: SEQ ID NO: 107, CDR2: SEQ ID NO: 108, CDR3: SEQ ID    NO: 109, VL: CDR1: SEQ ID NO: 110, CDR2: SEQ ID NO: 111, CDR3: SEQ    ID NO: 112;-   (xii) VH: CDR1: SEQ ID NO: 113, CDR2: SEQ ID NO: 114, CDR3: SEQ ID    NO: 115, VL: CDR1: SEQ ID NO: 116, CDR2: SEQ ID NO: 117, CDR3: SEQ    ID NO: 118;-   (xiii) VH: CDR1: SEQ ID NO: 119, CDR2: SEQ ID NO: 120, CDR3: SEQ ID    NO: 121, VL: CDR1: SEQ ID NO: 122, CDR2: SEQ ID NO: 123, CDR3: SEQ    ID NO: 124;-   (xiv) VH: CDR1: SEQ ID NO: 125, CDR2: SEQ ID NO: 126, CDR3: SEQ ID    NO: 127, VL: CDR1: SEQ ID NO: 128, CDR2: SEQ ID NO: 129, CDR3: SEQ    ID NO: 130;-   (xv) VH: CDR1: SEQ ID NO: 131, CDR2: SEQ ID NO: 132, CDR3: SEQ ID    NO: 133, VL: CDR1: SEQ ID NO: 134, CDR2: SEQ ID NO: 135, CDR3: SEQ    ID NO: 136;-   (xvi) VH: CDR1: SEQ ID NO: 137, CDR2: SEQ ID NO: 138, CDR3: SEQ ID    NO: 139, VL: CDR1: SEQ ID NO: 140, CDR2: SEQ ID NO: 141, CDR3: SEQ    ID NO: 142;-   (xvii) VH: CDR1: SEQ ID NO: 143, CDR2: SEQ ID NO: 144, CDR3: SEQ ID    NO: 145, VL: CDR1: SEQ ID NO: 146, CDR2: SEQ ID NO: 147, CDR3: SEQ    ID NO: 148;-   (xviii) VH: CDR1: SEQ ID NO: 149, CDR2: SEQ ID NO: 150, CDR3: SEQ ID    NO: 151, VL: CDR1: SEQ ID NO: 152, CDR2: SEQ ID NO: 153, CDR3: SEQ    ID NO: 154;-   (xix) VH: CDR1: SEQ ID NO: 155, CDR2: SEQ ID NO: 156, CDR3: SEQ ID    NO: 157, VL: CDR1: SEQ ID NO: 158, CDR2: SEQ ID NO: 159, CDR3: SEQ    ID NO: 160;-   (xx) VH: CDR1: SEQ ID NO: 161, CDR2: SEQ ID NO: 162, CDR3: SEQ ID    NO: 163, VL: CDR1: SEQ ID NO: 164, CDR2: SEQ ID NO: 165, CDR3: SEQ    ID NO: 166;-   (xxi) VH: CDR1: SEQ ID NO: 167, CDR2: SEQ ID NO: 168, CDR3: SEQ ID    NO: 169, VL: CDR1: SEQ ID NO: 170, CDR2: SEQ ID NO: 171, CDR3: SEQ    ID NO: 172;-   (xxii) VH: CDR1: SEQ ID NO: 173, CDR2: SEQ ID NO: 174, CDR3: SEQ ID    NO: 175, VL: CDR1: SEQ ID NO: 176, CDR2: SEQ ID NO: 177, CDR3: SEQ    ID NO: 178; and-   (xxiii) VH: CDR1: SEQ ID NO: 179, CDR2: SEQ ID NO: 180, CDR3: SEQ ID    NO: 181, VL: CDR1: SEQ ID NO: 182, CDR2: SEQ ID NO: 183, CDR3: SEQ    ID NO: 184.

In an embodiment fully human anti-CLDN18.2 antibodies, or antigenbinding fragments thereof, comprise a VH comprising a set ofcomplementarity-determining regions CDR1, CDR2, and CDR3 selected fromthe group consisting of:

-   (i) CDR1: SEQ ID NO: 221, CDR2: SEQ ID NO: 222, CDR3: SEQ ID NO:    223;-   (ii) CDR1: SEQ ID NO: 227, CDR2: SEQ ID NO: 228, CDR3: SEQ ID NO:    229;-   (iii) CDR1: SEQ ID NO: 233, CDR2: SEQ ID NO: 234, CDR3: SEQ ID NO:    235;-   (iv) CDR1: SEQ ID NO: 239, CDR2: SEQ ID NO: 240, CDR3: SEQ ID NO:    241;-   (v) CDR1: SEQ ID NO: 245, CDR2: SEQ ID NO: 246, CDR3: SEQ ID NO:    247;-   (vi) CDR1: SEQ ID NO: 251, CDR2: SEQ ID NO: 252, CDR3: SEQ ID NO:    253;-   (vii) CDR1: SEQ ID NO: 257, CDR2: SEQ ID NO: 258, CDR3: SEQ ID NO:    259;-   (viii) CDR1: SEQ ID NO: 263, CDR2: SEQ ID NO: 264, CDR3: SEQ ID NO:    265;-   (ix) CDR1: SEQ ID NO: 269, CDR2: SEQ ID NO: 270, CDR3: SEQ ID NO:    271;-   (x) CDR1: SEQ ID NO: 275, CDR2: SEQ ID NO: 276, CDR3: SEQ ID NO:    277;-   (xi) CDR1: SEQ ID NO: 281, CDR2: SEQ ID NO: 282, CDR3: SEQ ID NO:    283;-   (xii) CDR1: SEQ ID NO: 287, CDR2: SEQ ID NO: 288, CDR3: SEQ ID NO:    289;-   (xiii) CDR1: SEQ ID NO: 293, CDR2: SEQ ID NO: 294, CDR3: SEQ ID NO:    295;-   (xiv) CDR1: SEQ ID NO: 299, CDR2: SEQ ID NO: 300, CDR3: SEQ ID NO:    301;-   (xv) CDR1: SEQ ID NO: 311, CDR2: SEQ ID NO: 312, CDR3: SEQ ID NO:    313;-   (xvi) CDR1: SEQ ID NO: 317, CDR2: SEQ ID NO: 318, CDR3: SEQ ID NO:    319; and-   (xvii) CDR1: SEQ ID NO: 323, CDR2: SEQ ID NO: 324, CDR3: SEQ ID NO:    325.

In an embodiment, fully human anti-CLDN18.2 antibodies, or antigenbinding fragments thereof, comprise a VL comprising a set ofcomplementarity-determining regions CDR1, CDR2, and CDR3 selected fromthe group consisting of:

-   (i) CDR1: SEQ ID NO: 224, CDR2: SEQ ID NO: 225, CDR3: SEQ ID NO:    226;-   (ii) CDR1: SEQ ID NO: 230, CDR2: SEQ ID NO: 231, CDR3: SEQ ID NO:    232;-   (iii) CDR1: SEQ ID NO: 236, CDR2: SEQ ID NO: 237, CDR3: SEQ ID NO:    238;-   (iv) CDR1: SEQ ID NO: 242, CDR2: SEQ ID NO: 243, CDR3: SEQ ID NO:    244;-   (v) CDR1: SEQ ID NO: 248, CDR2: SEQ ID NO: 249, CDR3: SEQ ID NO:    250;-   (vi) CDR1: SEQ ID NO: 254, CDR2: SEQ ID NO: 255, CDR3: SEQ ID NO:    256;-   (vii) CDR1: SEQ ID NO: 260, CDR2: SEQ ID NO: 261, CDR3: SEQ ID NO:    262;-   (viii) CDR1: SEQ ID NO: 266, CDR2: SEQ ID NO: 267, CDR3: SEQ ID NO:    268;-   (ix) CDR1: SEQ ID NO: 272, CDR2: SEQ ID NO: 273, CDR3: SEQ ID NO:    274;-   (x) CDR1: SEQ ID NO: 278, CDR2: SEQ ID NO: 279, CDR3: SEQ ID NO:    280;-   (xi) CDR1: SEQ ID NO: 284, CDR2: SEQ ID NO: 285, CDR3: SEQ ID NO:    286;-   (xii) CDR1: SEQ ID NO: 290, CDR2: SEQ ID NO: 291, CDR3: SEQ ID NO:    292;-   (xiii) CDR1: SEQ ID NO: 296, CDR2: SEQ ID NO: 297, CDR3: SEQ ID NO:    298;-   (xiv) CDR1: SEQ ID NO: 302, CDR2: SEQ ID NO: 303, CDR3: SEQ ID NO:    304;-   (xv) CDR1: SEQ ID NO: 314, CDR2: SEQ ID NO: 315, CDR3: SEQ ID NO:    316;-   (xvi) CDR1: SEQ ID NO: 320, CDR2: SEQ ID NO: 321, CDR3: SEQ ID NO:    322; and-   (xvii) CDR1: SEQ ID NO: 326, CDR2: SEQ ID NO: 327, CDR3: SEQ ID NO:    328.

SEQ ID NOs and the amino acid sequences of the variable domains (VH andVL) and CDRs of the murine anti-CLDN18.2 antibodies are provided in FIG.1.

SEQ ID NOs and the amino acid sequences of the variable domains (VH andVL) and CDRs of the human anti-CLDN18.2 antibodies are provided in FIG.2.

In an embodiment, antibodies having the ability of binding to CLDN18.2comprise a combination of VH and VL each comprising a set ofcomplementarity-determining regions CDR1, CDR2 and CDR3 selected fromthe group consisting of:

-   (i) VH: CDR1: SEQ ID NO: 221, CDR2: SEQ ID NO: 222, CDR3: SEQ ID NO:    223, VL: CDR1: SEQ ID NO: 224, CDR2: SEQ ID NO: 225, CDR3: SEQ ID    NO: 226;-   (ii) VH: CDR1: SEQ ID NO: 227, CDR2: SEQ ID NO: 228, CDR3: SEQ ID    NO: 229, VL: CDR1: SEQ ID NO: 230, CDR2: SEQ ID NO: 231, CDR3: SEQ    ID NO: 232;-   (iii) VH: CDR1: SEQ ID NO: 233, CDR2: SEQ ID NO: 234, CDR3: SEQ ID    NO: 235, VL: CDR1: SEQ ID NO: 236, CDR2: SEQ ID NO: 237, CDR3: SEQ    ID NO: 238;-   (iv) VH: CDR1: SEQ ID NO: 239, CDR2: SEQ ID NO: 240, CDR3: SEQ ID    NO: 241, VL: CDR1: SEQ ID NO: 242, CDR2: SEQ ID NO: 243, CDR3: SEQ    ID NO: 244;-   (v) VH: CDR1: SEQ ID NO: 245, CDR2: SEQ ID NO: 246, CDR3: SEQ ID NO:    247, VL: CDR1: SEQ ID NO: 248, CDR2: SEQ ID NO: 249, CDR3: SEQ ID    NO: 250;-   (vi) VH: CDR1: SEQ ID NO: 251, CDR2: SEQ ID NO: 252, CDR3: SEQ ID    NO: 253, VL: CDR1: SEQ ID NO: 254, CDR2: SEQ ID NO: 255, CDR3: SEQ    ID NO: 256;-   (vii) VH: CDR1: SEQ ID NO: 257, CDR2: SEQ ID NO: 258, CDR3: SEQ ID    NO: 259, VL: CDR1: SEQ ID NO: 260, CDR2: SEQ ID NO: 261, CDR3: SEQ    ID NO: 262;-   (viii) VH: CDR1: SEQ ID NO: 263, CDR2: SEQ ID NO: 264, CDR3: SEQ ID    NO: 265, VL: CDR1: SEQ ID NO: 266, CDR2: SEQ ID NO: 267, CDR3: SEQ    ID NO: 268;-   (ix) VH: CDR1: SEQ ID NO: 269, CDR2: SEQ ID NO: 270, CDR3: SEQ ID    NO: 271, VL: CDR1: SEQ ID NO: 272, CDR2: SEQ ID NO: 273, CDR3: SEQ    ID NO: 274;-   (x) VH: CDR1: SEQ ID NO: 275, CDR2: SEQ ID NO: 276, CDR3: SEQ ID    NO: 277. VL: CDR1: SEQ ID NO: 278, CDR2: SEQ ID NO: 279, CDR3: SEQ    ID NO: 280;-   (xi) VH: CDR1: SEQ ID NO: 281, CDR2: SEQ ID NO: 282, CDR3: SEQ ID    NO: 283, VL: CDR1: SEQ ID NO: 284, CDR2: SEQ ID NO: 285, CDR3: SEQ    ID NO: 286;-   (xii) VH: CDR1: SEQ ID NO: 287, CDR2: SEQ ID NO: 288, CDR3: SEQ ID    NO: 289, VL: CDR1: SEQ ID NO: 290, CDR2: SEQ ID NO: 291, CDR3: SEQ    ID NO: 292;-   (xiii) VH: CDR1: SEQ ID NO: 293, CDR2: SEQ ID NO: 294, CDR3: SEQ ID    NO: 295, VL: CDR1: SEQ ID NO: 296, CDR2: SEQ ID NO: 297, CDR3: SEQ    ID NO: 298;-   (xiv) VH: CDR1: SEQ ID NO: 299, CDR2: SEQ ID NO: 300, CDR3: SEQ ID    NO: 301, VL: CDR1: SEQ ID NO: 302, CDR2: SEQ ID NO: 303, CDR3: SEQ    ID NO: 304;-   (xv) VH: CDR1: SEQ ID NO: 311, CDR2: SEQ ID NO: 312, CDR3: SEQ ID    NO: 313, VL: CDR1: SEQ ID NO: 314, CDR2: SEQ ID NO: 315, CDR3: SEQ    ID NO: 316;-   (xvi) VH: CDR1: SEQ ID NO: 317, CDR2: SEQ ID NO: 318, CDR3: SEQ ID    NO: 319, VL: CDR1: SEQ ID NO: 320, CDR2: SEQ ID NO: 321, CDR3: SEQ    ID NO: 322; and-   (xvii) VH: CDR1: SEQ ID NO: 323, CDR2: SEQ ID NO: 324, CDR3: SEQ ID    NO: 325, VL: CDR1: SEQ ID NO: 326, CDR2: SEQ ID NO: 327, CDR3: SEQ    ID NO: 328.

In an embodiments, the anti-CLDN18.2 antibodies or antibody fragmentsthereof comprise a variable heavy chain sequence selected from the groupconsisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 215, 217, and 219 and/or avariable light chain sequence selected from the group consisting of: SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 216, 218, and 220.

In an embodiment, the anti-CLDN18.2 antibody or antigen binding fragmentthereof comprises a specific pair of variable heavy chain and variablelight chain sequences, selected from the following combinations: avariable heavy chain sequence comprising SEQ ID NO: 1 and a variablelight chain sequence comprising SEQ ID NO: 2; a variable heavy chainsequence comprising SEQ ID NO: 3 and a variable light chain sequencecomprising SEQ ID NO: 4; a variable heavy chain sequence comprising SEQID NO: 5 and a variable light chain sequence comprising SEQ ID NO: 6; avariable heavy chain sequence comprising SEQ ID NO: 7 and a variablelight chain sequence comprising SEQ ID NO: 8; a variable heavy chainsequence comprising SEQ ID NO: 9 and a variable light chain sequencecomprising SEQ ID NO: 10; a variable heavy chain sequence comprising SEQID NO: 11 and a variable light chain sequence comprising SEQ ID NO: 12;a variable heavy chain sequence comprising SEQ ID NO: 13 and a variablelight chain sequence comprising SEQ ID NO: 14; a variable heavy chainsequence comprising SEQ ID NO: 15 and a variable light chain sequencecomprising SEQ ID NO: 16; a variable heavy chain sequence comprising SEQID NO: 17 and a variable light chain sequence comprising SEQ ID NO: 18;a variable heavy chain sequence comprising SEQ ID NO: 19 and a variablelight chain sequence comprising SEQ ID NO: 20; a variable heavy chainsequence comprising SEQ ID NO: 21 and a variable light chain sequencecomprising SEQ ID NO: 22; a variable heavy chain sequence comprising SEQID NO: 23 and a variable light chain sequence comprising SEQ ID NO: 24;a variable heavy chain sequence comprising SEQ ID NO: 25 and a variablelight chain sequence comprising SEQ ID NO: 26; a variable heavy chainsequence comprising SEQ ID NO: 27 and a variable light chain sequencecomprising SEQ ID NO: 28; a variable heavy chain sequence comprising SEQID NO: 29 and a variable light chain sequence comprising SEQ ID NO: 30;a variable heavy chain sequence comprising SEQ ID NO: 31 and a variablelight chain sequence comprising SEQ ID NO: 32; a variable heavy chainsequence comprising SEQ ID NO: 33 and a variable light chain sequencecomprising SEQ ID NO: 34; a variable heavy chain sequence comprising SEQID NO: 35 and a variable light chain sequence comprising SEQ ID NO: 36;a variable heavy chain sequence comprising SEQ ID NO: 37 and a variablelight chain sequence comprising SEQ ID NO: 38; a variable heavy chainsequence comprising SEQ ID NO: 39 and a variable light chain sequencecomprising SEQ ID NO: 40; a variable heavy chain sequence comprising SEQID NO: 41 and a variable light chain sequence comprising SEQ ID NO: 42;a variable heavy chain sequence comprising SEQ ID NO: 43 and a variablelight chain sequence comprising SEQ ID NO:44; and a variable heavy chainsequence comprising SEQ ID NO:45 and a variable light chain sequencecomprising SEQ ID NO:46.

In an alternative embodiment, fully human anti-CLDN18.2 antibodies, orantigen binding fragment thereof, comprise a specific pair of variableheavy chain and variable light chain sequences, selected from thefollowing combinations: a variable heavy chain sequence comprising SEQID NO: 185 and a variable light chain sequence comprising SEQ ID NO:186; a variable heavy chain sequence comprising SEQ ID NO: 187 and avariable light chain sequence comprising SEQ ID NO: 188; a variableheavy chain sequence comprising SEQ ID NO: 189 and a variable lightchain sequence comprising SEQ ID NO: 190; a variable heavy chainsequence comprising SEQ ID NO: 191 and a variable light chain sequencecomprising SEQ ID NO: 192; a variable heavy chain sequence comprisingSEQ ID NO: 193 and a variable light chain sequence comprising SEQ ID NO:194; a variable heavy chain sequence comprising SEQ ID NO: 195 and avariable light chain sequence comprising SEQ ID NO: 196; a variableheavy chain sequence comprising SEQ ID NO: 197 and a variable lightchain sequence comprising SEQ ID NO: 198; a variable heavy chainsequence comprising SEQ ID NO: 199 and a variable light chain sequencecomprising SEQ ID NO: 200; a variable heavy chain sequence comprisingSEQ ID NO: 201 and a variable light chain sequence comprising SEQ ID NO:202; a variable heavy chain sequence comprising SEQ ID NO: 203 and avariable light chain sequence comprising SEQ ID NO: 204; a variableheavy chain sequence comprising SEQ ID NO: 205 and a variable lightchain sequence comprising SEQ ID NO: 206; a variable heavy chainsequence comprising SEQ ID NO: 207 and a variable light chain sequencecomprising SEQ ID NO: 208; a variable heavy chain sequence comprisingSEQ ID NO: 209 and a variable light chain sequence comprising SEQ ID NO:210; a variable heavy chain sequence comprising SEQ ID NO: 211 and avariable light chain sequence comprising SEQ ID NO: 212; a variableheavy chain sequence comprising SEQ ID NO: 215 and a variable lightchain sequence comprising SEQ ID NO: 216; a variable heavy chainsequence comprising SEQ ID NO: 217 and a variable light chain sequencecomprising SEQ ID NO: 218; and a variable heavy chain sequencecomprising SEQ ID NO: 219 and a variable light chain sequence comprisingSEQ ID NO: 220.

In an alternative embodiment, the anti-CLDN18.2 antibodies or antibodyfragments thereof comprise a pair of variable heavy chain and variablelight chain sequences, selected from the following combinations: avariable heavy chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 1 and a variable light chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 2; a variable heavy chain sequence that is 90%,95%, or 99% identical to SEQ ID NO: 3 and a variable light chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 4; a variableheavy chain sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 5and a variable light chain sequence that is 90%, 95%, or 99% identicalto SEQ ID NO: 6; a variable heavy chain sequence that is 90%, 95%, or99% identical to SEQ ID NO: 7 and a variable light chain sequence thatis 90%, 95%, or 99% identical to SEQ ID NO: 8; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 9 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 10; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 11 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 12; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 13 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 14; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 15 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 16; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 17 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 18; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 19 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 20; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 21 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 22; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 23 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 24; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 25 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 26; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 27 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 28; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 29 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 30; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 31 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 32; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 33 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 34; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 35 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 36; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 37 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 38; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 39 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 40; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 41 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 42; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 43 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 44; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 45 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 46.

In another alternative embodiment, the anti-CLDN18.2 antibodies orantibody fragments thereof comprise a pair of variable heavy chain andvariable light chain sequences, selected from the followingcombinations: a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 185 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 186; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 187 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 188; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 189 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 190; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 191 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 192; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 193 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 194; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 195 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 196; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 197 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 198; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 199 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 200; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 201 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 202; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 203 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 204; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 205 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 206; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 207 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 208; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 209 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 210; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 211 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 212; a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 215 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 216; a variable heavy chainsequence that is 90%, 95%, or 99% identical to SEQ ID NO: 217 and avariable light chain sequence that is 90%, 95%, or 99% identical to SEQID NO: 218;. a variable heavy chain sequence that is 90%, 95%, or 99%identical to SEQ ID NO: 219 and a variable light chain sequence that is90%, 95%, or 99% identical to SEQ ID NO: 220.

In some embodiments, the antibody is a full-length antibody. In otherembodiments, the antibody is an antibody fragment including, forexample, an antibody fragment selected from the group consisting of:Fab, Fab′, F(ab)2, Fv, domain antibodies (dAbs), and complementaritydetermining region (CDR) fragments, single-chain antibodies (scFv),chimeric antibodies, diabodies, triabodies, tetrabodies, miniantibodies,multispecific antibodies, bispecific antibodies and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer CLDN18.2-specific binding to the polypeptide.

Thus, in one embodiment, the antibody fragment comprises at least oneCDR as described herein. The antibody fragment may comprise at leasttwo, three, four, five, or six CDRs as described herein. The antibodyfragment further may comprise at least one variable region domain of anantibody described herein. The variable region domain may be of any sizeor amino acid composition and will generally comprise at least one CDRsequence responsible for specifically binding to human CLDN18.2, forexample, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 asdescribed herein, and which is adjacent to or in frame with one or moreframework sequences.

In some embodiments, the anti-CLDN8.2 antibody is a monoclonal antibody.In some embodiments, the anti-CLDN8.2 antibody is a human antibody. Inalternative embodiments, the anti-CLDN8.2 antibody is a murine antibody.In some embodiments, the anti-CLDN8.2 antibody is a chimeric antibody, abispecific antibody, or a humanized antibody.

In some embodiments, the anti-CLDN8.2 antibodies or antibody fragmentsthereof comprise one or more conservative amino acid substitutions. Aperson of skill in the art will recognize that a conservative amino acidsubstitution is a substitution of one amino acid with another amino acidthat has similar structural or chemical properties, such as, forexample, a similar side chain. Exemplary conservative substitutions aredescribed in the art, for example, in Watson et al., Molecular Biologyof the Gene, The Benjamin/Cummings Publication Company, 4th Ed. (1987).

“Conservative modifications” refer to amino acid modifications that donot significantly affect or alter the binding characteristics of theantibody containing the amino acid sequences. Conservative modificationsinclude amino acid substitutions, additions and deletions. Conservativesubstitutions are those in which the amino acid is replaced with anamino acid residue having a similar side chain. The families of aminoacid residues having similar side chains are well defined and includeamino acids with acidic side chains (e.g., aspartic acid, glutamicacid), basic side chains (e.g., lysine, arginine, histidine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), uncharged polar side chains (e.g., glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine,tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan,histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine,valine, leucine, isoleucine, serine, threonine), amide (e.g.,asparagine, glutamine), beta-branched side chains (e.g., threonine,valine, isoleucine) and sulfur-containing side chains (cysteine,methionine). Furthermore, any native residue in the polypeptide may alsobe substituted with alanine, as has been previously described foralanine scanning mutagenesis (MacLennan et al. (1998) Acta Physiol ScandSuppl 643: 55-67; Sasaki et al. (1998) Adv Biophys 35: 1-24). Amino acidsubstitutions to the antibodies of the invention may be made by knownmethods for example by PCR mutagenesis (U.S. Pat. No. 4,683,195).

In one embodiment, the IL-CLDN18.2 antibody or antibody fragment thereofcomprises all six of the CDR regions of the Ms1, Ms2, Ms3, Ms4, Ms5,Ms6, Ms7, Ms8, Ms9, Ms10, Ms11, Ms12, Ms13, Ms14, Ms15, Ms16, Ms17,Ms18, Ms19, Ms20, Ms21, Ms22 or, Ms23 antibodies formatted as a chimericor a humanized antibody. In other embodiments, the CLDN18.2 antibody orantibody fragment thereof comprises all six of the CDR regions of one ofthe disclosed fully human antibodies.

In some embodiments, a variable region domain of an anti-CLDN18.2antibody disclosed herein may be covalently attached at a C-terminalamino acid to at least one other antibody domain or a fragment thereof.Thus, for example, a VH domain that is present in the variable regiondomain may be linked to an immunoglobulin CH1 domain, or a fragmentthereof. Similarly, a VL domain may be linked to a CK domain or afragment thereof. In this way, for example, the antibody may be a Fabfragment wherein the antigen binding domain contains associated VH andVL domains covalently linked at their C-termini to a CH1 and CK domain,respectively. The CH1 domain may be extended with further amino acids,for example, to provide a hinge region or a portion of a hinge regiondomain as found in a Fab fragment, or to provide further domains, suchas antibody CH2 and CH3 domains.

Antigen binding fragments, monospecific or multispecific antibodies maybe produced by recombinant DNA techniques or by enzymatic or chemicalcleavage of intact antibodies. Examples of antibody fragments includebut are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab)2; diabodies; linearantibodies; single-chain antibody molecules (e.g., scFv). “Single-chainFv” also abbreviated as “sFv” or “scFv” are antibody fragments thatcomprise the VH and VL antibody domains connected into a singlepolypeptide chain. One or more of the disclosed anti-CLDN18 binding CDRsmay be incorporated into a recombinant molecule either covalently ornoncovalently to make it an antigen binding protein in the format ofchimeric antibodies, diabodies, triabodies, tetrabodies, andpolypeptides that contain at least a portion of an immunoglobulin thatis sufficient to confer CLDN18 specific binding to the polypeptide.

Thus, in one embodiment, the binding agent comprises at least one CDR asdescribed herein. The binding agent may comprise at least two, three,four, five, or six CDRs as described herein. The binding agent furthermay comprise at least one variable region domain of an antibodydescribed herein. The variable region domain may be of any size or aminoacid composition and will generally comprise at least one CDR sequenceresponsible for binding to human CLDN18.2, for example, CDR-H1, CDR-H2,CDR-H3 and/or the light chain CDRs specifically described herein andwhich is adjacent to or in frame with one or more framework sequences.

In general terms, the variable (V) region domain may be any suitablearrangement of immunoglobulin heavy (VH) and/or light (VL) chainvariable domains. Thus, for example, the V region domain may bemonomeric and be a VH or VL domain, which is capable of independentlybinding human CLDN18.2 as described below. Alternatively, the V regiondomain may be dimeric and contain VH-VH, VH-VL, or VL-VL, dimers. The Vregion dimer comprises at least one VH and at least one VL chain thatmay be non-covalently associated (hereinafter referred to as FV). Ifdesired, the chains may be covalently coupled either directly, forexample via a disulfide bond between the two variable domains, orthrough a linker, for example, a peptide linker, to form a single-chainFv (scFV).

A person of skill in the art will recognize that a human IgGimmunoglobulin molecule (antibody) consists of four polypeptide chains,composed of two identical 50 kDa γ heavy (H) chains and two identical 25kDa κ or λ light (L) chains, linked together by inter-chain disulfidebonds. Each heavy chain consists of an N-terminal variable domain (VH)and an Fc region comprising three constant domains (CH1, CH2, CH3), withan additional “hinge region” between CH1 and CH2. Each light chainconsists of an N-terminal variable domain (VL) and a single constantdomain (CL). Two types of light chains, kappa (κ) and lambda (λ), werealso originally defined serologically, and subsequently by protein andgene sequences. Each H2L2 module expresses either two kappa or twolambda light chains to form H2κ2 or H2λ2 heterodimers.

The Fc-region of IgG heavy chain molecules also contains a bindingepitope, located at the interface between the Fc CH2-CH3 domains for theneonatal Fc receptor (FcRn) that is responsible for placental transportand IgG half-life. Generally, the variable regions of an antibody (VHand VL domain) are involved in the recognition of an epitope on a targetand the Fc fragment functions to confer the biological properties of theantibody. More specifically, the Fc region allows the antibody to berecognized by the immune effectors (including monocytes, macrophages,dendritic cells, and natural killer or NK cells) expressing Fcγreceptors, to activate the complement system, and to bind to the FcRn(neonatal Fc receptor).

Human IgG comprises four highly conserved subclasses: IgG1, IgG2, IgG3and IgG1 4 that show over 90% homology in amino acid sequence, withsubtype-specific differences that affect their binding to accessorymolecules and receptors. The subclasses of IgG differ from each other inthe sequence of their constant region, particularly in the hinge andupper CH2 domains that are involved in binding to both IgG-Fc receptors(Fc y receptors) and the complement component C1q. As a result, thedifferent IgG subclasses have different effector functions, both interms of activating immune effector cells and activating the complementcascade. The types of FcγR-mediated effector cell responses include, butare not limited to, antibody-dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), the release ofcytokines and antigen uptake for presentation.

In addition to isotypic variation, allelic variation is also found amongthe IgG subclasses. These polymorphic epitopes of immunoglobulins thatcan differ between individuals and ethnic groups were originallydiscovered on the basis of serological findings, as immunogenicdeterminants found on IgG from some individuals but not others. Theallotypes are inherited in a codominant Mendelian way, and various setsof combinations are found in African, white, and Oriental populations.

One of the most commonly used sub-classes of the IgG isotype fortherapeutic antibodies is an IgG1 , which has four well-characterizedallotypes. It has been reported that the use of IgG1 sequencescomprising alternative allotypes can modify (increase or decrease) thebinding affinity for FcRn receptor, and/or increases the stability ofthe complex formed by the modified antibody and FcRn, thereby affectingthe pharmacokinetics of a therapeutic antibody (US2018/0362624, and, J.Immunol. 196(2):607 (2016), Ternant, D et al). Currently, IgG1 mAbtherapeutics of the four predominant allotypes are licensed and in themarketplace. It is noted that those of ordinary skill in the art canreadily determine a suitable IgG1 CH sequence for use with the VHregions of the invention.

In one aspect, the anti-CLDN18.2 antibodies provided herein comprise aspecific pair of the variable heavy chain and variable light chainsequences in combination with a wild-type human IgG1 heavy chainconstant region (CH) and wild-type human kappa light chain (CL) region.For example, and not for the purpose of limitation, the inventionprovides anti-CLDN18.2 antibodies comprising a heavy chain variableregion (VH) of the invention in combination with a wild-type human IgG1constant heavy chain (CH) immunoglobulin sequence selected from thesequences set forth in SEQ ID NOS: 331-334, and a wild-type humanimmunoglobulin light chain having a light chain variable region of theinvention (VL) in combination with an immunoglobulin light chainconstant region (CL). In some aspects, the CL region comprises a humankappa light chain region comprising the sequence set forth in SEQ ID NO:335. FIGS. 11A and 11B provide sequence information for human IgG1 CHdomains (FIG. 11A) and a human kappa chain domain (FIG. 11B) suitablefor use in combination with the VH and VL sequences of the invention.

In another aspect, the anti-CLDN18.2 antibodies provided herein comprisea human IgG1 CH region comprising a modified CH that includes pointmutations introduced into the sequence to enhance a desirable effectorfunction such as ADCC or ADCP activity or to prolong the half-life orstability of the antibody. In one aspect the invention provides ananti-CLDN18.2 antibody engineered or manufactured in a manner intendedto make it “fit-for-purpose” as a cancer immunotherapy due to anenhanced ADCC, ADCP and/or CDC activity. More specifically, one aspectof the invention features an anti-CLDN18.2 antibody characterized by lowor no IgG-Fc core fucose. Potential advantages of a non-fucosylatedanti-CLDN18.2 therapeutic antibody include the potential of achievingtherapeutic efficacy at lower doses, inducing high cellular cytotoxicityagainst tumor cells that express low levels of CLDN18.2, and triggeringhigh effector function in NK cells expressing CD16a low-affinity Fcγreceptors.

In one aspect, and not for the purpose of limitation, the inventionfeatures an anti-CLDN18.2 antibody characterized by low or no IgG corefucose (non- or afucosylated) antibodies. One of skill in the art willreadily appreciate that this aspect of the invention may be a chimericor humanized anti-CLDN18.2 antibody comprising variable regions (i.e.,VH and VL) regions selected from the pairs of antibody sequences (VH andVL) or the sets of VH and VL CDRs, in FIG. 1. Alternatively, theanti-CLDN18.2 antibody characterized by low or no IgG core fucose can bea fully human sequence comprising variable regions selected from thepairs of antibody sequences (VH and VL) or the sets of VH and VL CDR inFIG. 2.

In one aspect, the antibodies of the invention feature non-fucosylatedanti-CLDN18.2 antibodies, or anti-CLDN18.2 antibodies characterized bylow core IgG fucose levels, comprising a pair of VH and VL sequencesselected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ IDNO:4, SEQ ID NO: 201 and SEQ ID NO: 202, and SEQ ID NO: 203 and SEQ IDNO: 204, respectively. In any particular antibody, the anti-CLDN18.2specific variable regions can be in the format of a full-length IgGcomprising a human IgG1 constant heavy chain immunoglobulin sequenceselected from SEQ ID NOS: 331-334, and a human Ig kappa light chainconstant region comprising the sequence set forth in SEQ ID NO: 335.

In an alternative aspect, the invention features a CLDN18.2 antibodycomprising point mutations at targeted sites in the Fc region to enhanceADCC, and/or ADCP and/or CDC activities relative to the activity of thesame antibody (e.g., same variable VH and VL regions or set of 6 set CDRsequences) comprising a wild-type human IgG1 CH sequence. One of skillin the art will readily appreciate that this aspect of the invention maybe a chimeric or humanized anti-CLDN18.2 antibody comprising variableregions (i.e., VH and VL) regions selected from the pairs of antibodysequences (VH and VL) or the sets of VH and VL CDRs, in FIG. 1.Alternatively, the Fc-engineered anti-CLDN18.2 antibody comprising pointmutations can be a fully human sequence comprising variable regionsselected from the pairs of antibody sequences (VH and VL) or the sets ofVH and VL CDR in FIG. 2.

In one aspect, the invention provides Fc-engineered anti-CLDN18.2specific antibodies comprising a pair of VH and VL sequences selectedfrom SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4, SEQ IDNO: 201 and SEQ ID NO: 202, and SEQ ID NO: 203 and SEQ ID NO: 204,respectively. In any particular antibody, the anti-CLDN18.2 specificvariable regions can be in the format of a full length IgG comprising ahuman IgG1 constant heavy chain immunoglobulin sequence selected fromSEQ ID NOS: 331-334, and a human Ig kappa light chain constant regioncomprising the sequence set forth in SEQ ID NO: 335.

For example, and not for the purpose of limitation, the inventionprovides an Fc engineered fully human IgG1 anti-CLDN18.2 antibodycomprising the VH sequence provided in SEQ ID NO: 201 combined with anengineered CH region that includes point mutations in the CH1 and/or CH2and/or CH3 region designed to enhance a desirable biological activity;and a VL sequence provided in SEQ ID NO: 202 combined with a human kappalight chain immunoglobulin sequence, such as the sequence set forth inSEQ ID NO: 335. In one aspect, and not for the purpose of limitation,the invention features an anti-CLDN18.2 antibody, referred to herein asmAb NBL-014, comprising a recombinant heavy chain having the amino acidsequence SEQ ID NO: 336 and a light chain comprising the sequence setforth in SEQ ID NO: 337 (see FIG. 12). In an alternative embodiment, theinvention provides an anti-CLDN18.2 antibody comprising a recombinantheavy chain having the amino acid sequence SEQ ID NO: 338 and a lightchain comprising the sequence set forth in SEQ ID NO: 337, referred toherein as NBL-014 (see FIG. 12).

In an alternative aspect, the invention provides an Fc engineered fullyhuman IgG1 anti-CLDN18.2 antibody comprising the VH sequence provided inSEQ ID NO: 203 combined with an engineered CH region that includes pointmutations in the CH1 and/or CH2 and/or CH3 region designed to enhance adesirable biological activity; and a VL sequence provided in SEQ ID NO:204 combined with a human kappa light chain immunoglobulin sequence,such as the sequence set forth in SEQ ID NO: 335.

In an alternative aspect, the invention provides Fc-modified,glycoengineered or protein-engineered (e.g. by the introduction of pointmutations in the CH Fc region), anti-CLDN18.2 specific antibodiescomprising a VH sequence that includes CDR1, CDR2 and CDR3 regionsselected from the sets of VH and VL CDRs provided in FIGS. 1 and 2. Morespecifically the invention features engineered anti-CLDN18.2 antibodiesa VH domain comprising a set of CDR1, CDR2, and CDR3 sequences selectedfrom SEQ ID NO:S 47-49, SEQ ID NOS: 53-55, SEQ ID NOS: 269-271, and SEQID NOS: 275-277, combined with an engineered CH region that includespoint mutations in the CH1 and/or CH2 and/or CH3 region designed toenhance a desirable biological activity, paired with a VL sequencecomprising a set of CDR1, CDR2 and CDR3 sequences selected from SEQ IDNOS: 50-52, SEQ ID NOS: 56-58, SEQ ID NOS: 272-274, and SEQ ID NOS:278-280 respectively, combined with a human kappa light chainimmunoglobulin sequence.

The therapeutic value of the antibodies of the disclosure can beenhanced by conjugation to a cytotoxic drug or agent that improves itseffectiveness and potency. In some embodiments the antibody is anantibody drug conjugate (ADC) comprising a CLDN18.2-specific antibodycoupled to a cytotoxic effector agent such as a radioisotope, a drug, ora cytotoxin. Internalization by endocytosis indicates that an antibodyis suitable for antibody drug conjugate (ADC) development. Anti-CLDN18.2antibodies having these properties can also be applied, but are notlimited to bispecific, chimeric antigen receptor T or NK cells, celltherapy, and combinational therapies for treating CLDN18.2-associateddiseases or disorders.

The anti-CLDN18.2 antibodies of the disclosure can also be used fordeveloping antibody-based immunotherapeutics that rely onCLDN18.2-specific binding to direct patient effector cells (e.g., Tcells or NK cells) to tumors including bispecific T cell engagingantibodies, or bispecific molecules that redirect NK cells, or celltherapies, such as CAR-T therapy.

Methods of Producing Antibodies

Many suitable methods can be applied for antibody generation and areknown to those of skill in the art. For example, a recipient may beimmunized with cells that express claudin 18.2, soluble recombinantclaudin 18.2 protein, or a fragment or a peptide conjugated with acarrier protein thereof. Any suitable method known to those of skill inthe art may be used to elicit an antibody with desired biologicproperties to inhibit claudin 18.2. Such methods can include a method ofimmunization that includes the use of adjuvants, other immunestimulants, repeat booster immunizations, and the use of one or moreimmunization routes.

Any suitable source of claudin 18.2 can be used as the immunogen for thegeneration of the non-human antibody or human antibody, specific forclaudin 18.2 of the compositions and methods disclosed herein. Suchforms include, but are not limited to cells expressing claudin 18.2(endogenous' cells or cells that are transfected with claudin 18.2 gene)whole protein, peptide(s), and epitopes, generated through recombinant,synthetic, chemical or enzymatic degradation means known in the art.

Different forms of the antigens may be used to generate the antibodythat is sufficient to generate a biologically active antibody. Thus, theeliciting antigen may be a single epitope, multiple epitopes, or theentire protein alone or in combination with one or more immunogenicityenhancing agents. In some cases, the eliciting antigen is an isolatedfull-length protein, a cell surface protein (e.g., immunizing with cellstransfected with at least a portion of the antigen), or a solubleprotein (e.g., immunizing with only the extracellular domain portion ofthe protein).

In some embodiments, the antigen is produced in a genetically modifiedcell. The DNA encoding the antigen may be genomic or non-genomic (e.g.,cDNA), and may encode at least a portion of the extracellular domain. Asused herein, the term “portion” refers to the minimal number of aminoacids or nucleic acids, as appropriate, to constitute an immunogenicepitope of the antigen of interest. Any genetic vectors suitable fortransformation of the cells of interest may be employed, including, butnot limited to adenoviral vectors, plasmids, and non-viral vectors, suchas cationic lipids.

It is desirable to prepare monoclonal antibodies (mAbs) from variousmammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Sties et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY(4^(th) ed.) Lance Medical Publication, Los Altos, Calif., andreferences cited therein; Harlow and Lane (1988) ANTIBODIES: ALABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE (2D ed) Academic Press, New York, N.Y.Typically, spleen cells from an animal immunized with a desired antigenare immortalized, commonly by fusion with a myeloma cell. See Kohler andMilstein (196) Eur. J. Immunol. 6:511-519. Alternative methods ofimmortalization include transformation with Epstein Barr Virus,oncogene, or retroviruses, or other methods known in the art. See. e.g.,Doyle et al. (eds. 1994 and periodic supplements) CELL AND TISSUECULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, N.Y.Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity for theantigen, and yield of the monoclonal antibodies produced by such cellsmay be enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. Alternatively, one may isolateDNA sequences which encode a monoclonal antibody or an antigen bindingfragment thereof by screening a DNA library from human B cellsaccording, e.g., to the general protocol outlined by Huse et al. (1989)Science 246: 1275-1281. Thus, monoclonal antibodies may be obtained by avariety of techniques familiar to researchers skilled in the art.

Other suitable techniques involve selection of libraries of antibodiesin phage, yeast, virus or similar vector. See e.g., Huse et al. supra;and Ward et al. (1989) Nature 341:544-546. The polypeptides andantibodies of the present invention may be used with or withoutmodification, including chimeric or humanized antibodies. Frequently,the polypeptides and antibodies will be labeled by joining, eithercovalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties,magnetic particles, and the like. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,9396,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly U.S. Patent No. 4,816,567; and Queen et al.(1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10023; or made in transgenicmice, see Nils Lonberg et al. (1994), Nature 368:856-859; and Mendez etal. (1997) Nature Genetics 15: 146-156; TRANSGENIC ANIMALS AND METHODSOF USE (WO2012US62118A), Medarex, Trianni, Abgenix, Ablexis, OminiAb,Harbour and other technologies.

In some embodiments, the ability of the produced antibody to bind anantigen (e.g., CLDN18) can be assessed using standard binding assays,such as ELISA, Western Blot, Immunofluorescent and flow cytometricanalysis. In some aspects, the produced antibody may also be assessedfor its ability to mediate the killing of target cells (e.g. tumorcells) expressing CLDN18 (e.g., antibody-dependent cell-mediatedcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC),antibody-dependent phagocytosis (ADCP)and/or inhibition of cellproliferation).

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a typical purification technique. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human gamma1, gamma2, orgamma4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth.62:1-13). Protein G is recommended for all mouse isotypes and for humangamma3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575). A matrix towhich an affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, reverse phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, typically performed at low salt concentrations(e.g., from about 0-0.25M salt).

Also included are nucleic acids that hybridize under low, moderate, andhigh stringency conditions, as defined herein, to all or a portion(e.g., the portion encoding the variable region) of the nucleotidesequence represented by isolated polynucleotide sequence(s) that encodean antibody or antibody fragment of the present disclosure. Thehybridizing portion of the hybridizing nucleic acid is typically atleast 15 (e.g., 20, 25, 30 or 50) nucleotides in length. The hybridizingportion of the hybridizing nucleic acid is at least 80%, e.g., at least90%, at least 95%, or at least 98%, identical to the sequence of aportion or all of a nucleic acid encoding an anti-CLDN18 polypeptide(e.g., a heavy chain or light chain variable region), or its complement.Hybridizing nucleic acids of the type described herein can be used, forexample, as a cloning probe, a primer, e.g., a PCR primer, or adiagnostic probe.

Polynucleotides, Vectors, and Host Cells

Other embodiments encompass isolated polynucleotides that comprise asequence encoding an anti-CLDN18.2 antibody or antibody fragmentthereof, vectors, and host cells comprising the polynucleotides, andrecombinant techniques for production of the antibody. The isolatedpolynucleotides can encode any desired form of the anti-CLDN18.2antibody including, for example, full-length monoclonal antibodies, Fab,Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments.

Some embodiments include isolated polynucleotides comprising sequencesthat encode the heavy chain variable region of an antibody or antibodyfragment having the amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,215, 217, or 219. Some embodiments include isolated polynucleotidescomprising sequences that encode the light chain variable region of anantibody or antibody fragment having the amino acid sequence of any ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 216, 218, or 220.

In an embodiment, the isolated polynucleotide sequence(s) encodes anantibody or antibody fragment having a light chain and a heavy chainvariable region comprising the amino acid sequences of:

-   (a) a variable heavy chain sequence comprising SEQ ID NO: 1 and a    variable light chain sequence comprising SEQ ID NO: 2;-   (b) a variable heavy chain sequence comprising SEQ ID NO: 3 and a    variable light chain sequence comprising SEQ ID NO: 4;-   (c) a variable heavy chain sequence comprising SEQ ID NO: 5 and a    variable light chain sequence comprising SEQ ID NO: 6;-   (d) a variable heavy chain sequence comprising SEQ ID NO: 7 and a    variable light chain sequence comprising SEQ ID NO: 8;-   (e) a variable heavy chain sequence comprising SEQ ID NO: 9 and a    variable light chain sequence comprising SEQ ID NO: 10;-   (f) a variable heavy chain sequence comprising SEQ ID NO: 11 and a    variable light chain sequence comprising SEQ ID NO: 12;-   (g) a variable heavy chain sequence comprising SEQ ID NO: 13 and a    variable light chain sequence comprising SEQ ID NO: 14;-   (h) a variable heavy chain sequence comprising SEQ ID NO: 15 and a    variable light chain sequence comprising SEQ ID NO: 16;-   (i) a variable heavy chain sequence comprising SEQ ID NO: 17 and a    variable light chain sequence comprising SEQ ID NO: 18;-   (j) a variable heavy chain sequence comprising SEQ ID NO: 19 and a    variable light chain sequence comprising SEQ ID NO: 20;-   (k) a variable heavy chain sequence comprising SEQ ID NO: 21 and a    variable light chain sequence comprising SEQ ID NO: 22;-   (l) a variable heavy chain sequence comprising SEQ ID NO: 23 and a    variable light chain sequence comprising SEQ ID NO: 24;-   (m) a variable heavy chain sequence comprising SEQ ID NO: 25 and a    variable light chain sequence comprising SEQ ID NO: 26;-   (n) a variable heavy chain sequence comprising SEQ ID NO: 27 and a    variable light chain sequence comprising SEQ ID NO: 28;-   (o) a variable heavy chain sequence comprising SEQ ID NO: 29 and a    variable light chain sequence comprising SEQ ID NO: 30;-   (p) a variable heavy chain sequence comprising SEQ ID NO: 31 and a    variable light chain sequence comprising SEQ ID NO: 32;-   (q) a variable heavy chain sequence comprising SEQ ID NO: 33 and a    variable light chain sequence comprising SEQ ID NO: 34;-   (r) a variable heavy chain sequence comprising SEQ ID NO: 35 and a    variable light chain sequence comprising SEQ ID NO: 36;-   (s) a variable heavy chain sequence comprising SEQ ID NO: 37 and a    variable light chain sequence comprising SEQ ID NO: 38;-   (t) a variable heavy chain sequence comprising SEQ ID NO: 39 and a    variable light chain sequence comprising SEQ ID NO: 40;-   (u) a variable heavy chain sequence comprising SEQ ID NO: 41 and a    variable light chain sequence comprising SEQ ID NO: 42;-   (v) a variable heavy chain sequence comprising SEQ ID NO: 43 and a    variable light chain sequence comprising SEQ ID NO: 44; or-   (w) and a variable heavy chain sequence comprising SEQ ID NO: 45 and    a variable light chain sequence comprising SEQ ID NO: 46.

In another embodiment, the isolated polynucleotide sequence(s) encodesan antibody or antibody fragment having a light chain and a heavy chainvariable region comprising the amino acid sequences of:

-   (aa) a variable heavy chain sequence comprising SEQ ID NO: 185 and a    variable light chain sequence comprising SEQ ID NO: 186;-   (bb) a variable heavy chain sequence comprising SEQ ID NO: 187 and a    variable light chain sequence comprising SEQ ID NO: 188;-   (cc) a variable heavy chain sequence comprising SEQ ID NO: 189 and a    variable light chain sequence comprising SEQ ID NO: 190;-   (dd) a variable heavy chain sequence comprising SEQ ID NO: 191 and a    variable light chain sequence comprising SEQ ID NO: 192;-   (ee) a variable heavy chain sequence comprising SEQ ID NO: 193 and a    variable light chain sequence comprising SEQ ID NO: 194;-   (ff) a variable heavy chain sequence comprising SEQ ID NO: 195 and a    variable light chain sequence comprising SEQ ID NO: 196;-   (gg) a variable heavy chain sequence comprising SEQ ID NO: 197 and a    variable light chain sequence comprising SEQ ID NO: 198;-   (hh) a variable heavy chain sequence comprising SEQ ID NO: 199 and a    variable light chain sequence comprising SEQ ID NO: 200;-   (ii) a variable heavy chain sequence comprising SEQ ID NO: 201 and a    variable light chain sequence comprising SEQ ID NO: 202;-   (jj) a variable heavy chain sequence comprising SEQ ID NO: 203 and a    variable light chain sequence comprising SEQ ID NO: 204;-   (kk) a variable heavy chain sequence comprising SEQ ID NO: 205 and a    variable light chain sequence comprising SEQ ID NO: 206;-   (ll) a variable heavy chain sequence comprising SEQ ID NO: 207 and a    variable light chain sequence comprising SEQ ID NO: 208;-   (mm) a variable heavy chain sequence comprising SEQ ID NO: 209 and a    variable light chain sequence comprising SEQ ID NO: 210;-   (nn) a variable heavy chain sequence comprising SEQ ID NO: 211 and a    variable light chain sequence comprising SEQ ID NO: 212;-   (oo) a variable heavy chain sequence comprising SEQ ID NO: 215 and a    variable light chain sequence comprising SEQ ID NO: 216;-   (pp) a variable heavy chain sequence comprising SEQ ID NO: 217 and a    variable light chain sequence comprising SEQ ID NO: 218; or-   (qq) a variable heavy chain sequence comprising SEQ ID NO: 219 and a    variable light chain sequence comprising SEQ ID NO: 220.

In an alternative embodiment, the isolated polynucleotide sequence(s)encodes an antibody or antibody fragment having a light chain and aheavy chain variable region comprising the amino acid sequences of:

-   (aa) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 185 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 186;-   (bb) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 187 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 188;-   (cc) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 189 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 190;-   (dd) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 191 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 192;-   (ee) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 193 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 194;-   (ff) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 195 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 196;-   (gg) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 197 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 198;-   (hh) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 199 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 200;-   (ii) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 201 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 202;-   (jj) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 203 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 204;-   (kk) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 205 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 206;-   (ll) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 207 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 208;-   (mm) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 209 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 210;-   (nn) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 211 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 212;-   (oo) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 215 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 216;-   (pp) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 217 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 218; or-   (qq) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 219 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 220.

In an alternative embodiment, the isolated polynucleotide sequence(s)encodes an antibody or antibody fragment having a light chain and aheavy chain variable region comprising the amino acid sequences of:

-   (a) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 1 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 2;-   (b) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 3 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 4;-   (c) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 5 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 6;-   (d) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 7 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 8;-   (e) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 9 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 10;-   (f) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 11 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 12;-   (g) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 13 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 14;-   (h) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 15 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 16;-   (i) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 17 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 18;-   (j) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 19 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 20;-   (k) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 21 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 22;-   (l) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 23 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 24;-   (m) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 25 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 26;-   (n) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 27 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 28;-   (o) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 29 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 30;-   (p) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 31 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 32;-   (q) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 33 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 34;-   (r) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 35 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 36;-   (s) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 37 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 38;-   (t) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 39 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 40;-   (u) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 41 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 42;-   (v) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 43 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 44; or-   (w) a variable heavy chain sequence that is 90%, 95%, or 99%    identical to SEQ ID NO: 45 and a variable light chain sequence that    is 90%, 95%, or 99% identical to SEQ ID NO: 46.

The polynucleotide(s) that comprise a sequence encoding an anti-CLDN18.2antibody or antibody fragment thereof can be fused to one or moreregulatory or control sequence, as known in the art, and can becontained in suitable expression vectors or host cells as known in theart. Each of the polynucleotide molecules encoding the heavy or lightchain variable domains can be independently fused to a polynucleotidesequence encoding a constant domain, such as a human constant domain,enabling the production of intact antibodies. Alternatively,polynucleotides, or portions thereof, can be fused together, providing atemplate for production of a single-chain antibody.

For recombinant production, a polynucleotide encoding the antibody isinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Many suitable vectors for expressing the recombinantantibody are available. The vector components generally include but arenot limited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

The anti-CLDN18.2 antibodies or antibody fragments thereof can also beproduced as fusion polypeptides, in which the antibody or fragment isfused with a heterologous polypeptide, such as a signal sequence orother polypeptide having a specific cleavage site at the amino terminusof the mature protein or polypeptide. The heterologous signal sequenceselected is typically one that is recognized and processed (i.e.,cleaved by a signal peptidase) by the host cell. For prokaryotic hostcells that do not recognize and process the anti-CLDN18.2 antibodysignal sequence, the signal sequence can be substituted by a prokaryoticsignal sequence. The signal sequence can be, for example, alkalinephosphatase, penicillinase, lipoprotein, heat-stable enterotoxin IIleaders, and the like. For yeast secretion, the native signal sequencecan be substituted, for example, with a leader sequence obtained fromyeast invertase alpha-factor (including Saccharomyces and Kluyveromycesα-factor leaders), acid phosphatase, C. albicans glucoamylase, or thesignal described in WO90/13646. In mammalian cells, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, can be used. The DNA for such precursor region isligated in reading frame to DNA encoding the anti-CLDN18.2 antibody.

Expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors, this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2-v. plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV, andBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a gene that encodes aselectable marker to facilitate identification of expression. Typicalselectable marker genes encode proteins that confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, or alternatively, are complement auxotrophicdeficiencies, or in other alternatives supply specific nutrients thatare not present in complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

Antibody-Based Immunotherapy

The goal of antibody-based immunotherapy using tumor-antigen-targetingantibodies is to eliminate cancer cells without harming normal tissue.Therefore, the efficacy and safety of antibody-based immunotherapies inoncology vary depending in large part on the intended mechanism ofaction, the relevant effector function of the immune system and thenature of the tumor-associated target antigen. The optimal targetantigen should be accessible and expressed homogenously and exclusivelyon the surface of cancer cells. If the intended mechanism of action isantibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-mediated cytotoxicity (CDC), then in order to maximizeantibody Fc region interactions with components of the complement systemor immune effector cells, the antigen-mAb complex should not be rapidlyinternalized.

A critical feature of monoclonal antibodies is their high specificityand their ability to either directly reduce proliferation and/or induceapoptosis in target tumor cells, or to mark them for immune-effectormediated cell killing (complement-dependent cytotoxicity (CDC),antibody-dependent cellular cytotoxicity (ADCC)) (Kubota, T. et al.(2009) Cancer Sci. 100 (9), 1566-1572). Conjugation to cytotoxic drugscan expand the utility of monoclonal antibodies and improve theirpotency and effectiveness (Goldmacher, V. S. et al. (2011) Ther. Deliv.2 (3), 397-416; Sievers, E. L. (2013) Annu. Rev. Med. 64, 15-29).

Antibody-dependent cell-mediated cytotoxicity (ADCC) describes thecell-killing ability of effector cells, which preferably requires thetarget cell being marked by an antibody. Effector cells may include Bcells, T cells, killer cells, NK cells, macrophages, monocytes,eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mastcells, and/or basophils; more specifically effector cells are T cells orNK cells. In certain aspects, ADCC occurs when antibodies bind toantigens on tumor cells, and the antibody Fc domains engage Fc receptors(FcR) on the surface of immune effector cells. Several families of Fcreceptors have been identified, and specific cell populationscharacteristically express defined Fc receptors. ADCC can be viewed as amechanism to directly induce a variable degree of immediate tumordestruction that leads to antigen presentation and the induction oftumor-directed T-cell responses. Preferably, in vivo induction of ADCCwill lead to tumor-directed T-cell responses and host-derived antibodyresponses.

More specifically, there are six structurally distinct types of humanFcγ receptors (FcγRI or CD64, FcγRIIa/CD32a, FcγRIIb/CD32b,FcγRIIc/CD32c, FcγRIIIa/CD16a, and FcγRIIIb/CD16b) expressed onleukocytes of both the myeloid and lymphoid lineage. Fcγ receptors aredivided into two types: activating receptors (CD64, CD32a, CD32c, CD16a,and CD16b) that lead to immune cell activation through immunoreceptortyrosine-based activation motifs (ITAM) on cytosolic tails or onco-receptor molecules, and an inhibitory receptor (CD32b) that signalsthrough immunoreceptor tyrosine-based inhibitory motifs (ITIM).

Single-nucleotide polymorphisms (SNPs) in FCGR2A (H131R) and FCGR3A(V158F) are associated with improved outcomes attributed to a higherantibody binding affinity for human IgG1 and IgG2 therapeutic antibodieswhich results in increased ADCC activity (Vargas, F. A. et al, CancerCell 33: 649-663 (2018), Musolino, A. et al., J. Clin. Oncol. 26:1789-1796 (2008), Zhang, W. et al, J. Clin. Oncol. 25:3712-3718 (2007),Nordstrom, J. L. et al, Breast Cancer Res. 13:R123 (2011)). Theactivating CD16A FcγR occurs in two variants, or alleles, with high(158V) or low (158F) affinity for the Fc domain of IgG1 . A majority(approximately 85%) of the population carries the 158F genotype, eitherin the homozygous form or as heterozygous with 158V. Thus, FcgRgenotypes most frequently associated with greater beneficial responsesoccur in a minority of the population. This provides a strong rationalefor engineering the Fc domain of therapeutic antibodies developed forthe treatment of cancer to better interact with low-binding alleles ofactivating FcγRs to expand, without regard to FcγR genotype, with thegoal of developing therapeutics capable of conferring a benefit to agreater percentage of patients.

A critical step in the activation of cytotoxic cells is the binding ofmAbs to FcγRIIIa (CD16A) on immune effector cells, and the strength ofthis interaction is determined by antibody isotype, the glycosylationpattern of the antibody Fc region and FcγRIIIa polymorphisms. Numerouspublications have reported findings that demonstrate the role ofFcγR-mediated effector function in antibody-based cancer therapiesderived from clinical studies. The study results indicate an associationbetween clinical response (e.g., antibody efficacy) and specificalloforms of activating human FcγRs. Patients that carry the 158F allelehave been reported to show diminished clinical responses to certaintherapeutic antibodies, including trastuzumab, rituximab, cetuximab,infliximab and ipilimumab and other therapeutic antibodies that utilizeADCC as a major mechanism of action. Antibodies engineered to haveimproved FcgR binding profiles have been reported to drive superioranti-tumor responses and confer greater clinical benefit.

The discovery of activating and inhibitory FcγRs resulted intranslational research efforts focused on designing therapeuticantibodies that were “fit for purpose” based on having FcγR bindingactivities characterized by an activating/inhibiting (A:I) ratiodesigned to activate immune effector cells to perform particularfunctions. Immunotherapy of cancer with monoclonal antibodies (mAb)promotes the elimination of tumor cells by a variety of mechanismsincluding ADCC, ADCP and/or CDC activities. In practice, the therapeuticactivity of several approved mAbs depends on the binding of the Fcγregions to low-affinity Fcγ receptors expressed on effector cells.

Several publications report the successful use of protein engineeringstrategies to design variant human IgG1 Fc domains (CH regions) withoptimized FcgR binding profiles and activating/inhibiting (A: I) ratiossuitable to optimize cell-mediated effector functions. In particular,efforts have focused on increasing the affinity of the Fc domain for thelow-affinity receptor FcγIIIa. A number of mutations within the Fcdomain have been identified that either directly or indirectly enhancebinding of Fc receptors and as a result significantly enhance cellularcytotoxicity (Lazar, G. A. PNAS 103:4005-4010 (2006), Shields, R. L. etal, J. Biol. Chem. 276:6591-6604 (2001) Stewart, R. et al., ProteinEngineering Design and Selection 24: 671-678 (2011)). Researchers atGenentech identified the mutations S239D/A330L/I332E, MedImmuneidentified the mutation F243L (Stewart et al) and Xencor identifiedG236A (Richards, J. O. et al, Mol. Cancer Ther. 7:2517-2575 (2008)).

Several different companies, including, Xencor, Applied MolecularEvolution, Medimmune, Genentech and Macrogenics have described antibodyvariants, comprising at least one amino acid modification relative to awild-type Fc region, wherein the variant Fc region binds FcγRIIIA and/orFcγRIIA with a greater affinity, relative to a comparable moleculecomprising the wild-type Fc region. Xencor has developed the Xmabplatform technology and published results of IgG1 Fc variants comprisingS239D/I332E point mutations and S239D/A330L/I332E as optimized Fcvariants with desirable A:I ratios. Applied Molecular Evolution hasidentified IgG variants comprising P247I and A339Q point mutations.Macrogenics has described variant human IgG1 Fc regions comprisingmutations at V3051, F243L, R292P, Y300L, and P396L (variant 18)(Stavenhagen, J. B. et al, Cancer Res. 67:8882-8990 (2007)) or L235V,F243L, R292P, Y300L, and P396L (Nordstrom, J. L. et al, Breast CancerRes. 13:R123 (2011)).

Nordstrom et al. reported that response to trastuzumab (HERCEPTIN™) inmetastatic breast cancer correlates with the expression of the highbinding variant (158V) of the activating Fcγ receptor CD16A. Using achimeric anti-HER2 monoclonal antibody MGAH22 (with specificity andaffinity similar to trastuzumab) that comprised a human IgG1 Fc domainengineered for increased binding to both alleles of human CD16A,Nordstrom demonstrated that the engineered Fc domain conferred enhancedADCC against all HER2-positive tumor cells tested, including cellsresistant to trastuzumab's anti-proliferative activity or expressing lowHER2 levels. The greatest improvement occurs with effector cellsisolated from donors homozygous or heterozygous for CD16A-158F, thelow-binding allele.

The Fc variations incorporated into MGHA22 were incorporated into theclinical-stage antibody product candidate margetuximab which has beenengineered to have an increased ability to bind to the activating Fc-γreceptor (CD16A) and a decreased ability to bind to the Fc-γ inhibitoryreceptor CD32B on immune effector cells. Macrogenics has recentlypublished preliminary data from the first randomized Phase 3 study thatwas designed to examine the potential benefit of Fc modification and therole of Fc-gamma receptor genotypes on anti-HER2 antibody efficacy. TheSOPHIA study (NCT02492711) is a randomized, open-label Phase 3 clinicaltrial evaluating margetuximab plus chemotherapy compared to trastuzumabplus chemotherapy in patients with HER2-positive metastatic breastcancer. The study met its first sequential primary endpoint ofprogression-free survival (PFS). The median PFS of patients treated withmargetuximab and chemotherapy was 5.8 months compared to 4.9 months inpatients treated with trastuzumab and chemotherapy. At the time of theprimary PFS analysis, overall survival (OS) data based on 158 eventswere immature. The median OS at that time was prolonged by 1.7 months inpatients treated with margetuximab and chemotherapy compared to patientstreated with trastuzumab and chemotherapy. For the exploratorysubpopulation of patients carrying the CD16A 158F allele, the median OSwas prolonged by 6.8 months in the margetuximab arm compared to thetrastuzumab arm (Macrogenics Press Release).

Glycoengineering strategies have also been used to develop therapeuticantibodies with optimized effector functions tailored to the treatmentof cancer. It is known that FcγRs interact with the carbohydrates on theCH2 domain and that the composition of these glycans has a substantialeffect on effector function activity. A highly conserved glycan atposition 297 in the Fc-region infers structural changes to the Fc-regionrequired for binding to the Fcγ receptor. Subtle differences in theglycan composition at this site can affect the Fc-structure and may alsoalter the interaction with Fcγ R by direct contact. Perhaps the bestexample of this is non-fucosylated antibodies that exhibit increasedbinding to CD16A and exhibit greatly enhanced ADCC activity (Niwa, R etal, Clin. Cancer Res. 10:6248-6255 (2004), Ferrara, C. et al, J. Biol.Chem 281:5032-5036 (2006)).

The efficacy of therapeutic antibodies is critically dependent onappropriate posttranslational modifications. In particular,glycosylation of the antibody Fc is essential for Fc receptor-mediatedactivity. Among the effector functions of antibody therapeutics, ADCChas been identified clinically as an important mechanism of anti-cancerantibodies. It is well known in the art that the absence of IgG-Fc corefucose vastly increases binding to Fcγ RIIa (CD16a) and results inhigher ADCC potency (e.g., lower EC50), a finding that has beensuccessfully used to improve the efficacy of therapeutic antibodies.Glycoengineering strategies include producing antibodies underconditions that will provide low or no fucosylation (e.g.,non-fucosylated mAb) in order to enhance ADCC by increasing FcGRIIIabinding.

One of skill in the art will acknowledge that BioWa/Kyowa Hakko Kirinand GlycArt/Roche, have each developed proprietary cell lines that yielddefucosylated antibodies. To achieve this result, BioWa/Kyowa KirinHakko established α-1,6-fucosyltransferase (FUT8) enzyme knockoutChinese hamster ovary cell line (POTELLIGENT® technology), whileGlycArt/Roche chose to over-express heterologousβ-1,4-N-acetylglucosaminyltransferase III in antibody-producing cells(GlycoMab™ technology, U.S. Pat. No. 6,602,684).

Glycoengineered antibodies with improved ADCC activities have beenapproved by the Food and Drug Administration (FDA) and are currentlymarketed in the United States, including mogamulizumab (POTELIGEO™),obinutuzumab (GAZYVA™) and benralizumab (FASENRA™). Mogamulizumab is anafucosylated anti-CCR4 antibody that is produced in a cell line with amutation in the FUT8 (a-1,6-fucosyltransferase) gene using thePOTELLIGENT™ technology platform developed by Biowa. In mammals, mostcore-fucosylation on N-glycans is formed by α-1,6 linkage. FUT8 is theonly α-1,6 fucosyltransferase that catalyzes the transfer of fucoseresidues from GDP-fucose to the innermost GlcNAc of the tri-mannosylcore structure via the α-1,6 linkage. Benralizumab is an anti-IL5receptor antibody that is also manufactured using the POTELLIGENT™technology platform and has been reported to be capable of inducinga >1000-fold amplification of antibody-dependent cell mediated ADCC(Pelaia, C et al, BioMed Research Int., Article ID 4839230 (2018).

Obinutuzumab is a type II anti-CD20 mAb characterized by having a lowfucose content as a consequence of the addition of aB-1,4-N-acetylglucosaminyl-transferase (GnIII) gene in the productioncell line (Glycart GLYCOMAB™ technology). The modification has theeffect of adding bisecting N-acetylglucosamine (GlcNac) to the N-glycanstructures, the presence of which interferes with fucosylation leadingto the production of highly enriched bisected non-fucosylatedglycosylation variants. The glycoengineering strategy used tomanufacture obinutuzumab confers a unique mechanism of action thatrelies on enhanced ADCC and ADCP, with reduced reliance on the primarymechanism of action used by two other anti-CD20 antibodies (Rituximaband ofatumumab) that rely primarily on CDC (Tobinai, K et al, Adv. Ther.34(2): 324-356 (2017)). A person skilled in the art will also recognizethat it is possible to produce antibodies with low fucose content byusing a decoy substrate of glycosyltransferases, such as2-deoxy-2-fluoro-1-fucose (2FF,) or a fucosyltransferase inhibitor suchas 2F-peracetyl-fucose to cause reduced incorporation of fucose in theglycan of antibodies expressed in mammalian cells (Dekkers, G, et al,Scientific Reports 6, article number 36964 (2016)). It has been reportedthat antibodies produced using fucosyltransferase inhibitor showed asignificant decrease in the relative percentage of fucosylated glycanspecies (G0f, G1f, and G2f) and that the percentage of fucosylation canbe decreased from 86% to 19% in the presence of the inhibitor (Ho, D. etal. Fucosylation of a Therapeutic Antibody: Effects onAntibody-dependent Cell-mediated cytotoxicity (ADCC) Potency andEfficacy, Bioprocess International, Apr. 12, 2016).

Complement-dependent cytotoxicity (CDC) is another cell-killing methodthat can be directed by antibodies. IgM is the most effective isotypefor complement activation, but IgG1 and IgG3 are also both veryeffective at directing CDC via the classical complement activationpathway.

In some aspects, antibodies described herein are used in combinationwith or are conjugated to, a therapeutic moiety or agent, such as acytotoxin, a drug, or a radioisotope. In certain aspects, an antibodydescribed herein is used in combination with a chemotherapeutic agent.In certain aspects, an antibody-drug conjugate (ADC) comprises anantibody described herein conjugated (e.g., covalently attached) to atherapeutic moiety or agent.

Examples of therapeutic agents that may be used in conjugation with anantibody, include antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, fludarabin, 5-fluorouracil decarbazine),alkylating agents (e.g., mechlorethamine, thioepa chlorambucil,melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide,busulfan, dibromomannitol, streptozotocin, mitomycin C, andcis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine). In certain aspects, the therapeutic agent is a cytotoxicagent or a radiotoxic agent. In another aspect, the therapeutic agent isa photosensitizing agent, suitable for use in photodynamic therapy, suchas porfimer sodium. In some aspects, the therapeutic agent is animmunosuppressant. In some aspects, the therapeutic agent is GM-CSF. Insome aspects, the therapeutic agent is doxorubicin, cisplatin,bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide or ricinA.

Examples of a cytotoxin or cytotoxic agent include any agent that isdetrimental to and, in particular, kills cells, such as taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

In some aspects, an antibody described herein can be conjugated to aradioisotope, such as iodine-131, yttrium-90 or indium-111, to generatecytotoxic radiopharmaceuticals for treating a CLD18-related disorder,such as cancer.

Techniques for conjugating a therapeutic moiety to antibodies are wellknown to those of skill in the art, see, e.g., Arnon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62: 119-58 (1982).

In some embodiments, ADCs are designed to kill cancer cells in atarget-dependent manner. The first step in this process is the bindingof the antibody to its antigen. Upon ADC binding, the entire antigen-ADCcomplex is internalized and the cytotoxic payload is released into thetumor cell resulting in cell death. Factors that influence thetherapeutic index for ADCs include the tumor specificity of thetargeting antibody, the expression level of the tumor target antigen,the cytotoxic drug and the linker (Panowksi, S. et al. (2014) MAbs 6(1), 34-45). If the intended mechanism of action of a therapeuticantibody is downregulation of the tumor-associated antigen, or if theantibody is an ADC designed to deliver a toxin into the cancer cell theninternalization is a desirable characteristic of the antibody used todeliver the toxic payload to the targeted tumor cell.

In some aspects, an ADC comprises any agent that exerts a therapeuticeffect on cancer cells and an anti-CLDN18.2 antibody or derivativethereof as described herein. Conjugation of the drug does not alter orsignificantly alter the binding characteristics, in particular, thespecificity of the antibody. In certain aspects, the drug is a cytotoxicor cytostatic agent (e.g., any agent that is detrimental to and/or killscancer cells). Examples of classes of such cytotoxic agents includeanti-tubulin agents, DNA minor groove binders (e.g., enediynes andlexitropsins), DNA replication inhibitors, alkylating agents (e.g.,platinum complexes such as cis-platin, mono(platinum), bis(platinum) andtri-nuclear platinum complexes and carboplatin), anthracyclines,antibiotics, antifolates, antimetabolites, chemotherapy sensitizers,duocarmycins, etoposides, fluorinated pyrimidines, ionophores,nitrosoureas, platinols, pre-forming compounds, purine antimetabolites,puromycins, radiation sensitizers, steroids, taxanes (e.g., paclitaxeland docetaxel), topoisomerase inhibitors, vinca alkaloids, or the like.Additional examples of such cytotoxic agents are described in USPublication No. 2018/0117174.

The generation of antibody-drug conjugates can be accomplished by anytechnique known to the skilled artisan. Antibody-drug conjugates can beprepared by binding the drug to an antibody in accordance with aconventional technique. An antibody and a drug may be directly bound toeach other via their own linker groups or indirectly via a linker orother substance. Examples of linkers that may be used in forming an ADCare described in US Publication No. 2018/0117174.

One example of an anti-CLDN18.2 antibody being used in immunotherapy isiMAb362, a chimeric IgG1 monoclonal antibody, specific for an epitope inthe first extracellular domain of CLDN18.2. iMAb362, its murine parentalantibody, and other anti-CLDN18-specific antibodies are disclosed inpatents and patent applications belonging to the WO2007/059997,WO2014/075788, and WO2016/166122 patent families.

Preclinically, iMAb362 was shown to inhibit tumor growth and to killcancer cells by both indirect (complement-dependent cytotoxicity,antibody-dependent cellular cytotoxicity) and direct (antiproliferativeand proapoptotic effects) mechanisms of action. Astellas Pharma haspublished reports indicating that preclinical characterization ofiMAb362 antibody-drug conjugate (ADC) comprising the antimitotic drugmonomethyl auristatin E, with a valine-citrulline linker (iMAb-vcMMAE)is ongoing (Kreuzberg, M et al, Annals of Oncology 28(5) v122-v141,abstract 377P, WO2016/165762).

The phase IIb FAST trial enrolled patients with advanced or recurrentgastric or gastroesophageal junction adenocarcinoma (NCT01630083).Eligibility for enrollment required patients to have CLDN18.2-positivetumors (i.e., 2⁺/3⁺ intensity in ≥40% of tumor cells byimmunohistochemistry). The trial evaluated the role of iMAb362 incombination with chemotherapy versus chemotherapy alone in a first-linesetting. According to the final results, the addition of antibodyiMAB362 to chemotherapy (EOX, epirubicin, oxaliplatin and capecitabine)increased the median overall survival by five months (8.4 vs 13.2months, P=0.0001) in patients with advanced gastric, esophageal orgastroesophageal junction adenocarcinoma (Schuler, M et al., Annals ofOncology, 27 (6): vi207-vi242, abstract 6140 (2016)). The combinationtherapy also significantly improved progression-free survival andresponse rate. Reports indicate that when the results are stratified byCLDN18.2 expression levels, the best outcomes were seen among the highexpressers (≥2⁺ intensity in ≥70%) whose median overall survival was 9.0months vs. 16.7 months, P<0.0005). The results confirm that the additionof an anti-CLDN18.2 antibody to first-line chemotherapy provides aclinically relevant benefit in patients with advanced/recurrent gastriccancer.

Compositions and Methods of Treatment

The disclosure also provides compositions including, for example,pharmaceutical compositions that comprise an anti-CLDN18.2 antibody orantibody fragment thereof. Such compositions have numerous therapeuticuses for the treatment of diseases or disorders (e.g., diseases ordisorders involving cells expressing CLDN18.2). In some aspects, thecompositions described herein are administered to patients, e.g., invivo, to treat or prevent a disease associated with dysregulatedexpression of CLDN18.2. Preferred patients include human patients havingan epithelial cell-derived primary or metastatic cancer.

The antibodies can be administered either alone or in combination withother compositions that are useful for treating an immune-mediatedinflammatory disorder or an autoimmune disease. In some embodiments,compositions including, for example, pharmaceutical compositions,comprising the anti-CLDN18.2 antibody can further comprise a therapeuticagent, either conjugated or unconjugated to the anti-CLDN18.2 antibodyor antibody fragment.

In some aspects, the present invention provides a composition, e.g., apharmaceutical composition, containing one or more antibodies of thepresent invention. The pharmaceutical compositions may be formulatedwith pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients in accordance with conventionaltechniques such as those disclosed in Remington: The Science andPractice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co.,Easton, Pa., 1995. In some aspects, the pharmaceutical composition isadministered to a subject to treat cancer.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

Typically, compositions for administration by injection are solutions insterile isotonic aqueous buffer. Where necessary, the pharmaceutical canalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of the active agent. Where thepharmaceutical is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the pharmaceutical is administered by injection, anampoule of sterile water for injection or saline can be provided so thatthe ingredients can be mixed prior to administration.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid releases, such asa controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for the preparation of such formulations aregenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular subject, composition, and mode ofadministration, without being toxic to the subject. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical compositions described herein may be administered ineffective amounts. An “effective amount” refers to the amount whichachieves a desired reaction or the desired effect alone or together withfurther doses. In the case of treatment of a particular disease or of aparticular condition, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease.

In some aspects, the treatment of cancer represents a field wherecombination strategies are especially desirable since frequently thecombined action of two, three, four or even more cancer drugs/therapiesgenerates synergistic effects which are considerably stronger than theimpact of a monotherapeutic approach. The agents and compositions (e.g.,pharmaceutical compositions) provided herein may be used alone or incombination with conventional therapeutic regimens such as surgery,irradiation, chemotherapy and/or bone marrow transplantation(autologous, syngeneic, allogeneic or unrelated). The agents andcompositions may also be used in combination with one or more ofcytokines, chemokines, costimulatory molecules, or fusion proteins,bacterial treatments, kinase inhibitors, toll-like receptors,angiogenesis inhibitors, small molecule targeted therapy drugs,virus-based vaccines, multi-epitope strategies, adoptive T celltransfer, and peptide-based target therapies. See U.S. Pat. No.10,093,736. Thus, in another embodiment of the present invention, acancer treatment may be effectively combined with various other drugs.

In some aspects, the agents and compositions described herein areadministered to patients, e.g., in vivo, to treat or prevent a varietyof disorders such as those described herein. Preferred patients includehuman patients having disorders that can be corrected or ameliorated byadministering the agents and compositions described herein. Thisincludes disorders involving cells characterized by dysregulatedCLDN18.2 expression.

For example, in one embodiment, the agents and compositions describedherein can be used to treat a patient with a cancer disease, e.g., acancer disease such as described herein characterized by the presence ofcancer cells expressing CLDN18.2. In some aspects, a cancer disease isgastric cancer, esophageal cancer, pancreatic cancer, lung cancer,ovarian cancer, colon cancer, hepatic cancer, head-neck cancer, cancerof the gallbladder, or metastases thereof.

In an embodiment of the provided treatment method, the subject in needof therapy is also administered a therapeutic agent selected from thegroup consisting of an antineoplastic agent, a chemotherapeutic agent, agrowth inhibitory agent, a cytotoxic agent, and an immune checkpointinhibitor.

In an embodiment of the provided treatment methods, an antibody,antibody fragment, or bispecific antibody comprising an anti-CLDN18.2binding agent of the present invention is co-administered with achemotherapeutic agent, which may be a cytotoxic agent. For example,epitubicin, oxaliplatin, and/or 5-FU can be administered to patientsreceiving anti-Claudin 18.2 therapy.

The combination of therapeutic agents discussed herein can beadministered concurrently as components of a bispecific or multispecificbinding agent or fusion protein or as a single composition in apharmaceutically acceptable carrier. Alternatively, a combination oftherapeutics can be administered concurrently as separate compositionswith each agent in a pharmaceutically acceptable carrier. In anotherembodiment, the combination of therapeutic agents can be administeredsequentially.

In one embodiment, the treatment method of the invention furthercomprises administering an agent capable of stabilizing or increasingexpression of Claudin 18.2 at the cell surface of a cancer cell. Forexample, an agent stabilizing or increasing expression of Claudin 18.2may be oxaliplatin and/or 5-FU.

In some aspects, conventional viral and non-viral based gene transfermethods can be used to introduce nucleic acids encoding the antibodiesor derivatives thereof, as described herein, in mammalian cells ortarget tissues. Such methods can be used to administer nucleic acidsencoding the antibodies to cells in vitro. In some embodiments, thenucleic acids encoding the antibodies or derivatives thereof areadministered for in vivo or ex vivo gene therapy uses. In otherembodiments, gene delivery techniques are used to study the activity ofthe antibodies in cell-based or animal models. Non-viral vector deliverysystems include DNA plasmids, naked nucleic acid, and nucleic acidcomplexed with a delivery vehicle such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. Such methods are wellknown in the art.

Methods of non-viral delivery of nucleic acids encoding engineeredpolypeptides of the disclosure include lipofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection methods and lipofectionreagents are well known in the art (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids that are suitable for efficientreceptor-recognition lipofection of polynucleotides include those ofFeigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivoadministration) or target tissues (in vivo administration). Thepreparation of lipid: nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art.

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding the antibodies described herein take advantage of highlyevolved processes for targeting a virus to specific cells in the bodyand trafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of polypeptidesof the disclosure could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Viral vectors are currently the most efficient and versatile method ofgene transfer in target cells and tissues. Integration in the hostgenome is possible with the retrovirus, lentivirus, and adeno-associatedvirus gene transfer methods, often resulting in long term expression ofthe inserted transgene. Additionally, high transduction efficiencieshave been observed in many different cell types and target tissues.

All patents and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the disclosure. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents arebased on the information available to the applicants and do notconstitute any admission as to the correctness of the dates or contentsof these documents.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various embodiments hereindescribed and illustrated may be further modified to incorporatefeatures shown in any of the other embodiments disclosed herein.

The broad scope of this disclosure is best understood with reference tothe following examples, which are not intended to limit the disclosuresto the specific embodiments. The specific embodiments described hereinare offered by way of example only, and the disclosure is to be limitedby the terms of the appended claims, along with the full scope of theequivalents to which such claims are entitled.

EXAMPLES General Methods

Methods for protein purification including immunoprecipitation,chromatography, and electrophoresis are described. Coligan et al. (2000)Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc.,New York. Chemical analysis, chemical modification, post-translationalmodification, production of fusion proteins, and glycosylation ofproteins are described. See, e.g., Coligan et al. (2000) CurrentProtocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., NewYork; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol.3, John Wiley and Sons, Inc., NY, N.Y., pp. 16.0.5-16.22.17;Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis,Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory,Piscataway, N.J., pp. 384-391. Production, purification, andfragmentation of polyclonal and monoclonal antibodies are described.Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, JohnWiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlowand Lane, supra.

Hybridoma supernatant was purified via HiTrap protein G column (GE, cat.No. 17040401) according to the manufacturer's procedures. Briefly,hybridoma supernatant was equilibrated with DPBS (Gibco, cat. No.14190-136) for 5 CV and loaded via syringe/infusion pump (Legato 200,KDS) at ambient temperature and 3 minute residence time. The column waswashed with 5 CV of DPBS and elution was performed with 4 CV of pH 2.8elution buffer (Fisher Scientific, cat. No. PI21004). Elution wasfractionated, and fractions were neutralized with 1M Tris-HCL, pH 8.5(Fisher Scientific, cat No. 50-843-270) and assayed by A280(DropSense96, Trinean). Peak fractions were pooled, and buffer exchangedinto DPBS. Centrifugal filters (EMD Millipore, cat. No. UFC803024) wereequilibrated in DPBS at 4,000×g for 2 mins. Purified sample was loaded,DPBS was added and the sample was spun at 4,000×g for 5-10 minute spinsuntil total DPBS volume reached ≥6 DV. The final pool was analyzed byA280.

Standard methods in molecular biology are described. Maniatis et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif Standard methods also appear in Ausbel et al.(2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley andSons, Inc. New York, N.Y., which describes cloning in bacterial cellsand DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Stable cell lines expressing CLDN18.2 or CLDN18.1 were generated bytransfecting a selected host cell (i.e., CHO-K, NIH/3T3 or HEK293 cells,all purchased from ATCC) with pcDNA3.1-based plasmids expressing Homosapiens claudinl8, transcript variant 2 (CLDN18.2) or Homo sapiensclaudinl8, transcript variant 1 (CLDN18.1) using lipid-basedtransfection using Lipofectamine 3000 (Invitrogen, cat# L3000015).

Expression was confirmed using appropriate antibodies 24 h and 48 hafter transfection using flow cytometry to assay for surface expression.Antibiotic selection was used to select the integrated cell, after 7-10days of geneticin selection limiting dilution was performed on thesurviving cells in 96-well plate while keeping the transfectants underselection pressure (Geneticin).

After 10-14 days, single colonies were picked up for screening usingflow cytometry with CLDN18.2 or CLDN18.1-specific antibodies (CLDN18.2in-house version of a positive control antibody synthesized usingpublicly available sequence information for a monoclonal antibodyreported in US 2018/0117174 as being specific for CLDN18.2 (and notspecific for CLDN18.1) (referred to herein as a “positive control”antibody); CLDN18.1 Claudin 18 polyclonal antibody (Invitrogen, cat#38-8000)). The top 3-5 highly expressed clones were chosen for furtherdevelopment. After a couple of passages, the expression level wasconfirmed by flow cytometry and image assay to make sure it is stable.Specific gene expression was also confirmed by PCR.

The sequences for the heavy and light chain variable regions forhybridoma clones were determined as described below. Total RNA wasextracted from 1-2×10⁶ hybridoma cells using the RNeasy Plus Mini Kitfrom Qiagen (Germantown, Md., USA). cDNA was generated by performing 5′RACE reactions using the SMARTer RACE 5′/3′ Kit from Takara(Mountainview, Calif., USA). PCR was performed using the Q5High-Fidelity DNA Polymerase from NEB (Ipswitch, Mass., USA) to amplifythe variable regions from the heavy and light chains using the TakaraUniversal Primer Mix in combination with gene-specific primers for the3′ mouse constant region of the appropriate immunoglobulin. Theamplified variable regions for the heavy and light chains were run on 2%agarose gels, the appropriate bands excised and then gel purified usingthe Mini Elute Gel Extraction Kit from Qiagen. The purified PCR productswere cloned using the Zero Blunt PCR Cloning Kit from Invitrogen(Carlsbad, Calif., USA), transformed into Stellar Competent E. Colicells from Takara and plated onto LB Agar +50 ug/ml kanamycin plates.Direct colony Sanger sequencing was performed by GeneWiz (SouthPlainfield, N.J., USA). The resulting nucleotide sequences were analyzedusing IMGT V-QUEST to identify productive rearrangements and analyzetranslated protein sequences. CDR determination was based on IMGTnumbering.

Methods for flow cytometry, including fluorescence-activated cellsorting detection systems (FACS®), are available. See, e.g., Owens etal. (1994) Flow Cytometry Principles for Clinical Laboratory Practice,John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2nded.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry,John Wiley and Sons, Hoboken, N.J. Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable. Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

The in-house positive control CLDN18.2-specific antibody zolbetuximab(formerly designated claudiximab) referred to herein as “ PC1”, was madeby a CRO based on the public information for their VH and VL sequences(VH sequence: SEQ ID NO: 32 of US 2018/0117174; VL sequence: SEQ ID NO:39 of US 2018/0117174). The PCI antibody was used to confirm CLDN18.2expression by the transfectant and tumor cell lines used in the examplesand to establish the binding and functional assays used to evaluate andcharacterize the anti-CLDN18.2-specific antibodies disclosed herein.

Briefly, plasmids containing the control and benchmark antibodysequences were transfected using the ExpiCHO™ Expression System (CatalogNumber: A29133, ThermoFisher Scientific, USA) according to themanufacturer's protocol. The cells were cultured at 37° C. and 8% CO₂ onday 1 and then at 32° C. and 5% CO₂ post-transfection in media providedin the kit. Antibodies were purified by clarifying the ExpiCHO™ culturemedium by centrifugation at 1,000 g for 10 min followed by 5,000 g for30 min. The supernatant was then filtered using a 0.45 μητ filterfollowed by a 0.22 μητ filter. Subsequently, the supernatant wassubjected to affinity purification using protein A/G resins (LifeTechnologies, Carlsbad, Calif.; Catalog# 20424) according to themanufacturer's protocol. Prior to ELISA purification, antibody titer inthe culture medium was roughly determined to ensure the amount of mediumloaded occupied less than 80% of the resin binding capacity. Afterincubation, the resins were washed with PBS and eluted with ElutionBuffer (Life Technologies, Catalog# 21004). The elution fractions wereimmediately adjusted to physiologic pH by adding Tris Buffer, pH 8.0.The purified antibodies were subsequently subjected to buffer exchangeand protein concentration using Amicon Ultra-15 Centrifugal Filter Unit(Life Technologies, Catalog# UFC900324) in PBS buffer. Antibodyconcentration was determined by BCA Protein Assay. SDS-PAGE andCoomassie-staining were carried out to test the antibody purity. Thepurified protein was aliquoted and stored at −80° C. for long-timestorage or kept at 4° C. for immediate use.

The integrity of the antibody was validated by SDS-PAGE followed byCoomassie staining under non-reducing vs reducing conditions; undernon-reducing conditions, one dominating band around 150 kDa, whereasunder reducing conditions, two bands were observed, 50 kDa and 25 kDa.

Standard techniques for characterizing ligand/receptor interactions areavailable. See, e.g., Coligan et al. (2001) Current Protocols inImmunology, Vol. 4, John Wiley, Inc., New York. Standard methods ofantibody functional characterization appropriate for thecharacterization of antibodies with particular mechanisms of action arealso well known to those of skill in the art.

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains, CDRannotation, glycosylation sites, and sequence alignments, are available.

Example 1

Generation of Anti-CLDN18.2 Antibodies

Mouse anti-Claudin 18.2 antibodies were generated by immunizing Balb/cmice with HEK293 cells transfected with the human Claudin 18.2 gene.

Human anti-Claudin 18.2 antibodies were generated by immunizing human Igtransgenic mice, Trianni mouse strain (WO2013/063391) with HEK293 cellstransfected with human Claudin 18.2 gene.

Mice (Balb/c or human Ig transgenic) were immunized with eitherintraperitoneally (IP) or subcutaneously (SC). The immune response wasmonitored by retroorbital bleeds. The plasma was screened by FACS orImaging (as described below), and mice with sufficient titers ofanti-claudin 18.2 were used for fusions. Mice were boostedintraperitoneally or intravenously with the immunogens before sacrificeand removal of the spleen and lymph nodes.

To select Balb/c or human Ig transgenic mice producing antibodies thatbound Claudin 18.2, sera from immunized mice were screened by flowcytometry (FACS) for binding to a cell line expressing claudin 18.2(CHO, HEK293 or 3T3-claudin 18.2 cell lines) and not to control cellline that does not express claudin 18.2. Briefly, claudin 18.2-CHO (HEKor 3T3 cells) were incubated with dilutions of serum from immunizedmice. Cells were washed, and specific antibody binding was detected withPE-labeled anti-mouse IgG Ab. Flow cytometric analysis was performed ona flow cytometry instrument (Intellicyte, IQue plus, Sartorius). Inaddition, mice serum was tested by Imaging. Briefly, claudin 18.2-Cho(HEK or 3T3 cells) were incubated with dilutions of serum from immunizedmice. Cells were washed, fixed with paraformaldehyde, washed, specifiedantibody binding was detected with secondary Alexa488 goat anti-mouseantibody and Hoechst (Invitrogen, Calsbad, Calif.). Plates were scannedand analyzed on an imaging machine (Cytation 5, Biotek, Winooski, Vt.).Hybridoma supernatants were tested for anti-claudin 18.2 specificbinding by FACS and Imaging as described above.

To generate hybridomas producing human and mouse antibodies of theinvention, splenocytes and lymph node cells were isolated from animmunized mouse and fused to an appropriate immortalized cell line, suchas a mouse myeloma cell line. The resulting hybridomas were screened forthe production of antigen-specific antibodies. For example, single-cellsuspensions of splenocytes, lymph node cells from immunized mice werefused to the equal number of Sp2/0 non-secreting mouse IgG myeloma cells(ATCC, CRL 1581) by electrofusion. Cells were plated in flat-bottom96-well tissue culture plates, followed by 2 weeks of incubation in theselection medium (HAT medium), then switched to hybridoma culture media.Approximately 10-14 days after cell plating, supernatants fromindividual wells were screened by FACS or Imaging as described above.The antibody-secreting hybridomas were transferred to 24-well plates,screened again, and if still positive for anti-claudin 18.2, thepositive hybridomas were subcloned by limiting dilution or sorting usinga single cell sorter. The stable subclones were then cultured in vitroto generate small amounts of antibodies to be used for purification andfor characterization.

Example 2

Specificity of Anti-Claudin18.2 Antibodies

The binding specificity of the anti-claudin antibodies was evaluatedusing an immunofluorescence imaging assay.

The specific cellular binding affinity of anti-Claudin18.2 antibodieswas tested on CHO-CLDN18.2 (transfected CHO cell expressing recombinanthuman CLDN18.2) and CHO-CLDN18.1 (transfected CHO cell expressingrecombinant human CLDN18.1) cells. The cells were plated in completemedia containing F12 with 10% FBS, then incubated overnight at 37° C.,anti-Claudin18.2 antibodies were serial diluted and added to the assayplates, incubated at 4° C. for 2 hours, followed by fixing cells andfurther staining the cells for 30 minutes with Alexa Fluor® 488 GoatAnti-Human IgG (H+L) secondary antibody, invitrogen Cat#A-11013 fordetection. The binding was assessed by imaging the cells and quantifyingthe fluorescence intensity using a Biotek Cytation.

As demonstrated in FIG. 3A, the group of purified murine anti-Claudin18.2 antibodies binds to the CHO-CLDN18.2 cells, but not theCHO-CLDN18.1 cells when using 10 μg/ml of antibodies in the study, FIG.3B shows the representative data of dose-dependent binding of Ms-1, Ms-2and Ms-3 to CHO-CLDN18.2 cells with EC50 values of 1.2 nM, 2.8 nM, and1.8 nM respectively. The control Ab PC1 has an EC50 value of 3.2 nM,consistent with literature reports. The negative control antibodydoesn't bind to the CHO-CLDN18.2 cells.

The binding affinity of lead panel antibodies was also tested in a CHOCLDN18.1 cell line stably expressing Claudin18.1. None of the antibodiesshowed active binding activity at the highest testing concentration of133.4 nM. Data from representative clones are shown in FIG. 3C. Bothclones Ms-4 and Hu-3 exhibited dose-dependent binding to CHO-CLDN18.2cells with EC50 values of 0.75 nM and 1.34 nM, while not binding toCHO-CLDN18.1 cell.

The complete set of dose-dependent binding data of the murineanti-Claudin18.2 lead panel is summarized in Table 5. The group ofpurified murine anti-Claudin 18.2 antibodies binds to the CHO-CLDN18.2cells with EC50 values ranging from 0.8-3.3 nM.

TABLE 5 Specificity of Murine anti-CLDN18.2 Antibodies CHO-18.2 binding,PATU8988S mAb EC50 nM binding Ms1 1.2 +++ Ms2 3.0 ++ Ms3 1.8 +++ Ms4 0.8++++ Ms5 1.7 +++ Ms6 1.2 +++ Ms7 1.4 +++ Ms8 1.5 +++ Ms9 1.5 +++ Ms101.3 +++ Ms11 3.3 ++ Ms12 2.0 +++ Ms13 1.2 +++ Ms14 1.1 ++ Ms15 1.8 +++Ms16 0.8 ++++ Ms17 1.4 +++ Ms18 1.3 +++ Ms19 2.3 +++ Ms20 1.3 +++ Ms211.5 +++ Ms22 2.6 +++

Similarly, as shown in FIG. 4A, representative purified humananti-Claudin 18.2 antibodies bind to the CHO-CLDN18.2 cells, but not theCHO-CLDN18.1 cells when using 10 μg/ml of antibodies in the study.

The complete set of dose-dependent binding data of the humananti-Claudin18.2 lead panel is summarized in Table 6. The group of 17purified Claudin 18.2 antibodies binds to the CHO CLDN18.2 with EC50values ranging from 0.77 nM to 19 nM. Representative clone data isdemonstrated in FIG. 4B. Clones Hu-2, Hu-9, and Hu-10 exhibited bindingEC50 of 1.5, 1.1 and 1.5 nM.

TABLE 6 Specificity of Human anti-CLDN18.2 Antibodies CHO-18.2 PATU8988SmAb binding, EC50 nM binding Hu-1 3.6 ++ Hu-2 1.5 +++ Hu-3 1.3 +++ Hu-45.2 ++ Hu-5 4.5 + Hu-6 1.5 +++ Hu-7 1.0 ++++ Hu-8 3.7 ++ Hu-9 1.1 ++++Hu-10 1.5 ++++ Hu-11 17 ++ Hu-12 1.2 ++++ Hu-13 5.6 ++ Hu-14 19 + Hu-1612 + Hu-17 6.2 + Hu-18 0.8 +

Example 3

Binding Affinity of Claudin18.2 Antibodies to Tumor Cell LinesExpressing Claudin18.2

Target protein density and glycosylation status vary from cell type tocell type. The binding affinity and activity of affinity dependentmechanisms, such as ADCC and CDC, are significantly impacted by thetarget density and glycosylation status. Effects determined using celllines that endogenously express Claudin18.2 have more translationalrelevance to the efficacy of anti-Claudin18.2 antibodies.

Tumor cells were plated in complete media containing RPMI1640 with +10%FBS, then incubated overnight at 37° C., Claudin18.2 antibodies wereserial diluted and added to the assay plates, incubated at 4° C. for 2hours, followed by fixing cells and further staining the cells for 30minutes with Alexa Fluor® 488 Goat Anti-Human IgG (H+L) secondaryantibody (invitrogen Cat#A-11013) for detection. The binding wasassessed by imaging the cells and quantifying the fluorescence intensityusing a Biotek Cytation.

The relative expression level of different types of cells is provided inFIG. 5. Evaluation of the Claudin18.2 expression level in recombinantCHO-Claudin18.2 cells engineered to overexpress Claudin18.2 side by sidewith the gastric tumor cell line NUGC-4 and the pancreatic tumor cellline PATU8988S reveals that the CHO-Claudin18.2 expression level is morethan 100-fold higher than the endogenous expression level of NUGC-4 andPATU8988S cells.

The disclosed anti-CLDN18.2 antibodies demonstrate binding to thepancreatic tumor cell line PATU8988S (see Tables 5 and 6, above) withEC50 values ranging from less than 2 nM to about 100 nM. The strength ofthe binding are reported in Table 5 and Table 6 in four categories, thestrongest binding with EC50 values less than 2 nM are defined as “++++”,binding EC50 values between 2 nM and 20 nM are defined as “+++”, bindingEC50 values between 20 nM and 100 nM are marked as “++”, and weakbinding EC50 about 100 nM were indicated as “+”.

As shown in FIGS. 6A and 6B, the binding of anti-claudin18.2 antibodypanels exhibited better binding affinities to PATU8988S cells than thein-house positive control PC1 . When 20 μg/ml antibodies were used tostain the cells, the binding fluorescence intensity of PC1 issignificantly lower than the fluorescence intensity of Ms1-10 and Hu1-16antibodies.

Similarly, the binding affinity of the antibodies Hu-2, Hu-9 and Hu-10in the human antibody panel exhibited better binding affinities to thehuman gastric tumor cell line NUGC-4, shown by representative data inFIG. 7.

To further evaluate anti-claudin18.2 Ab binding affinity to tumor cells,another human gastric tumor cell line, KATOIII that endogenouslyexpresses claudin18.2 was used in a flow cytometry binding assay (FIG.8). Briefly, KatoIII cells were plated in complete media containingRPMI1640 with +10% FBS, Claudin18.2 antibodies were serial diluted andadded to the assay plates, incubated at 4° C. for 2 hours, followed byfixing cells and further staining the cells for 30 minutes with AlexaFluor® 488 Goat Anti-Human IgG (H+L) secondary antibody (InvitrogenCat#A-11013) for detection.

Antibody binding was assessed by flow cytometry instrument IQUE plus(Sartorius, Gottingen, Germany) . As shown in FIG. 8 , when 6.67 μg/mlantibodies were used to stain the cells, the binding fluorescenceintensity of the PC1 antibody in-house iMAb362 is significantly lowerthan the fluorescence intensity of Hu-2, Hu-9, and Hu-10 antibodies.

Example 4

Antibody-Dependent Cellular Cytotoxicity (ADCC)

Antibody-dependent cellular cytotoxicity (ADCC) of Claudin18.2antibodies bound to Claudin18.2 positive cells was measured by abioluminescence assay to determine the activity of the antibodies thatspecifically bind and activate mouse FcγRIV. Cells stably expressingCLDN18.2, either recombinant protein expressed by transfected CHO cellsor a human cell line expressing endogenous CLDN18.2, or negative cellswere plated in complete media containing F12+10%FBS, the cells wereincubated overnight at 37° C. Claudin18.2 antibodies were then serialdiluted and added together with effector cells. Cell viability wasdetected using a Promega bioluminescence assay following themanufacturer's instruction (Promega, cat#M1201).

As shown in Table 7, representative human Claudin 18.2 antibodies, whichhave human variable regions with mouse IgG2a Fc, induced ADCC with EC50values ranging from 0.77 nM to 7.09 nM. Representative data isdemonstrated in FIG. 9. Antibodies Hu-2, Hu-7, and Hu-9 exhibited ADCCEC50 of 3.51, 3.52, and 1.10 nM. An unrelated Mouse IgG2a antibody thatis not specific for CLDN18.2 shows no activity in the assay. The PC1antibody engineered to comprise a human IgG1 Fc, had an EC50 of 0.66 nMin a similar assay when adding human effector cells (Data not shown).

TABLE 7 Antibody dependent cellular cytotoxicity (ADCC) ActivityCHO-18.2 mAb EC50 nM Hu-1 0.77 Hu-2 3.51 Hu-3 3.11 Hu-4 2.39 Hu-5 7.09Hu-6 4.80 Hu-7 3.52 Hu-8 1.42 Hu-9 1.10 Hu-10 1.25 Hu-11 1.51 Hu-12 2.26Hu-13 1.17 Hu-14 1.51 Hu-16 3.09

Example 5

Endocytosis Screening

Endocytosis of the disclosed CLDN18.2-specific antibodies bound toClaudin18.2 positive cells was measured by a cytotoxicity basedendocytosis assay that used the co-internalization of the target boundantibody together with an anti-Human IgG Fc-MMAF Antibody.

Cells stably expressing Claudin 18.2 or negative cells were plated incomplete media containing F12+10% FBS, the cells were incubatedovernight at 37° C. Claudin18.2 antibodies were then serial diluted andadded together with anti-Human IgG Fc-MMAF antibody to the assay platesand incubated at 37° C. for 72 hours. Cell viability was detected usinga Promega Cell-titer Glo bioluminescence assay (Promega, cat#7570)following the manufacturer's instruction.

As demonstrated in Table 8, the lead panel of purified murine Claudin18.2 antibodies induced endocytosis derived cell toxicity in CHOCLDN18.2 cells with EC50 values ranging from 0.71-2.55 nM.

Similarly, as shown in Table 9, the Trianni panel of purified humanClaudin 18.2 antibodies induced endocytosis derived cell toxicity in CHOCLDN18.2 with EC50 values ranging from 0.85 nM to 13.39 nM.Representative clone data is demonstrated in FIG. 10. Clones Hu-2, Hu-7,Hu-9, and Hu-11 exhibited endocytosis derived cytotoxicity EC50 of 1.43,1.65, 0.85 and 1.31 nM, respectively.

The lead panel antibodies also exhibited endocytosis derived cellcytotoxicity in a pancreatic tumor cell line PATU8988S whichendogenously expresses Claudin18.2, but did not exhibit endocytosisderived cell killing activity in a CHO CLDN18.1 cell line stablyexpressing Claudin18.1 (data not shown).

TABLE 8 Endocytosis of Murine anti-Claudin18.2 mAbs CHO-18.2 Clone nameEC50 nM Ms1 1.09 Ms2 2.55 Ms3 1.14 Ms4 0.98 Ms5 1.43 Ms6 1.26 Ms7 1.12Ms8 1.08 Ms9 1.10 Ms10 0.96 Ms11 1.30 Ms12 1.07 Ms13 0.71 Ms14 1.02 Ms151.15 Ms16 1.00 Ms17 0.81 Ms18 1.39 Ms19 1.36 Ms20 1.07 Ms21 0.95 Ms221.61

TABLE 9 Endocytosis of Human anti-Claudin18.2 mAbs CHO-18.2 Clone nameEC50 nM Hu-1 1.61 Hu-2 1.43 Hu-3 1.23 Hu-4 1.55 Hu-5 13.39 Hu-6 1.69Hu-7 1.65 Hu-8 1.00 Hu-9 0.85 Hu-10 0.85 Hu-11 1.31 Hu-12 2.01 Hu-132.16 Hu-14 1.51 Hu-16 3.09

Example 6

Enhanced Antibody-Dependent Cellular Cytotoxicity (ADCC)

The ADCC activity of Claudin18.2 antibodies bound to Claudin18.2positive cells was measured by a bioluminescence assay to determine theactivity of the antibodies that specifically bind and activate humanFcγRIIIa. CHO cells stably expressing CLDN18.2, or negative cells, wereplated in complete media containing F12+10%FBS, the cells were incubatedovernight at 37° C. Claudin18.2 antibodies were then serial diluted andadded together with effector cells. Cell viability was detected using aPromega bioluminescence assay following the manufacturer's instruction(Promega, cat#7010) .

As shown in FIG. 13A, the unmodified anti-Claudin18.2 antibody NBL-014and the Fc modified antibodies NBL-014P, comprising point mutationsintroduced to enhance ADCC activity, and NBL-014G, an Fc-modifiedvariant produced in HEK cells under conditions selected to express anantibody characterized by low, or no IgG core fucose (e.g., non- orafucosylated), induced enhanced ADCC activity in CHO-claudin 18.2 cellsand PATU8988S cells.

In high target density CHO-claudin18.2 cells, NBL-014P and NBL-014Gexhibited EC50 values of 0.06 nM to 0.02 nM, 10-fold and 35-fold betterthan the unmodified parental NBL-014 antibody, which has an ADCC EC50 of0.73 nM (FIG. 13A).

An alternative ADCC assay was also used to evaluate the anti-claudin18.2antibody's ADCC activity by directly measuring the % target cell lysisusing effector cell line NK92MI-CD16a. Briefly, CHO-Claudin18.2 targetcells were stained with CFSE per manufacture's instruction, the cellswere then seeded into a U-bottom 384 well plate, at 800k/mL in RPMImedium (no antibiotics). Serial diluted Ab and isotype control wereadded into the plate. NK92MI cells were harvested and counted, anddiluted to indicated concentration, 320k/mL, then 10 ul/well was addedto have a final 1:5 ratio of NK92MI vs. tumor cells, incubated at 37°C., 5%CO₂ for 4 hours at 37° C. 5%CO₂, then taken out at room temp for10min. Dead cell dye 7-AAD (1:100) was added and mixed in the well,allowed to sit for 10 min, and the plate was then run on the FACS todetermine the cell death/killing ability (gate on CFSE positive todefine dead cell 7-AAD+).

As shown in FIG. 13B, the Fc modified antibodies NBL-014P and NBL-014Ginduced modestly enhanced ADCC activity in this assay format.

Example 7

Enhanced ADCC Activity in Tumor Cells Endogenously ExpressingClaudin18.2

Compared to CHO-claudin18.2 cells overexpressing Claudin18.2, the humantumor cell lines PATU8988S and NUGC-4 endogenously express much lessClaudin18.2 (FIG. 5). The anti-Claudin 18.2 antibodies were tested fortheir ADCC activity using the human tumor cell lines as target cells.The ADCC activity of Claudin18.2 antibodies was measured by abioluminescence assay to determine the activity of the antibodies thatspecifically bind and activate human FcγRIIIa. Human cell linesexpressing endogenous CLDN18.2 were plated in complete media containingRPMI1640+10%FBS, the cells were incubated overnight at 37° C.

Claudin18.2 antibodies were then serial diluted and added together witheffector cells. Cell viability was detected using a Promegabioluminescence assay following the manufacturer's instruction (Promega,cat#7010). In the PATU8988S cell line ADCC assay, NBL-014P and NBL-014Gshowed ADCC EC50 values of 0.06 nM, and 0.04 nM respectively, which areenhanced relative to the ADCC activity of parental clone NBL-014 (EC50of 0.12 nM, see FIG. 13A). Moreover, in this pancreatic cell model, themaximum of the ADCC effect upshifted ˜33 fold over the parental antibodyNBL-014's maximum ADCC response (FIG. 14B).

In NUGC-4 cell line, NBL-014P and NBL-014G showed ADCC EC50 of 0.04 nM,and 0.06 nM respectively, also enhanced from the parental clone NBL-014,which has an ADCC EC50 of 0.40 nM. Moreover, in this gastric cell assaythe maximum of the ADCC effect upshifted ˜3 fold over the parentalantibody NBL-014's maximum ADCC response (FIG. 14A).

Example 8

Complement-Dependent Cellular Cytotoxicity (CDC) of Anti-Claudin18.2Antibodies

The capability of anti-claudin18.2 antibodies to induce CDC activity wasfirst evaluated using the CHO cell line overexpressing claudin18.2. Thisflow cytometer-based CDC assay was done using pooled human serum fromPeproTech (Rockyhill, N.J.). CHO-Claudin18.2 cells were seeded in a384-well plate at 8,000 cells per well. 10 ul of selected antibodieswere added, at a serial dilution starting with a concentration of 10μg/ml. The plate was left to incubate for 15 minutes at 37° C. After theincubation, 10 ul of human serum was added to wells, with the finaldilution at 1/16th per volume. The plate was then left to incubate for2.5 hours at 37° C. 7AAD viability dye was added 10 minutes prior toiQue plus (flow cytometer) acquisition.

In high target density CHO-claudin18.2 cells, NBL-014P and NBL-014Gexhibited potent EC50 values of 0.15 nM to 0.30 nM, similar to itsun-engineered form NBL-014, which has a CDC EC50 of 0.22 nM (FIG.15).Relative to the CDC activity of PC1 (in-house iMAb36) (EC50 value of1.76), the observed EC50 activity of NBL-014 represents approximately a˜10 fold improvement.

The CDC activity of anti-claudin18.2 antibodies was further evaluated inthe human tumor cell lines PATU8988S endogenously expressingclaudin18.2. As Shown in FIG. 16, NBL-014, NBL-014P and NBL-014G allinduced strong CDC activity in PATU8988S, with NBL-014P having thehighest CDC % killing.

Example 9

Antibody-Dependent Cellular Phagocytosis (ADCP) of Anti-Claudin18.2Antibodies

The ability of anti-claudin18.2 antibodies to induce ADCP was assessedusing a Promega bioluminescence assay. The assay uses CHO cellsoverexpressing claudin18.2 or NUGC cells as target cells. The effectorcells were co-cultured with target cells in the presence ofanti-claudin18.2 antibodies. The antibody binds to the target on targetcells which in turn activate FcγRIIa on effector cells, resulting inNFAT-RE-mediated luciferase activity, that can be quantified using aBio-Glo Luciferase Assay system.

Briefly, CHO-claudin18.2 or NUGC-4 cells were plated in assay buffer inwhite 384-well plates, 12000/20 ul/well (0.6M/ml). The effector cellswere thawed and resuspended to 1M/ml and 10 ul was added to the assayplate (10000/well). Antibodies were diluted in assay buffer and added 10ul/well on top of the target cell. The cell plate was incubatedovernight. On the next day, 20 ul of luc substrate was directly addedaccording to manufacturer's instruction, the plate was spun down andread immediately.

The ADCP activity of anti-claudin18.2 antibodies NBL-014, NBL-014G andNBL-014P were shown in FIG. 17A (CHO-claudin18.2 cells) and FIG. 17B(NUGC-4 cells). When using a high-density CLDN18.2 target cell, theactivity from the Fc engineered Abs was similar to that of theirparental antibodies, while the ADCP activity of the in-house PCIantibody (iMAb362) exhibited a high response but less potent activity.

Use of the human tumor cell line NUGC-4 (endogenous expression ofCLDN18.2) to evaluate ADCP activity results in data indicating that thein-house PC1 antibody is 100 times less potent, but that its maximumactivity is higher than NBL-014, NBL-014P and NBL-014G.

Based on these results, it appears that the ADCP activity of NBL-014 ispotent regardless of the Claudin18.2 expression level, and that neitherof the Fc modifications (protein-engineering or glycol-engineering)changed the potency of the observed activity. The EC50 values ofNBL-014G and NBL-014P are similar to their parent clone NBL-014. In bothcell lines tested, the CHO-Claudin18.2 and NUGC-4, NBL-14, NBL-14G andNBL-14P demonstrated better ADCP activity in terms of EC50 values thanPC1. However, the ADCP effect at high antibody concentration of 10 μg/mlof PC1 has two-fold higher response than NBL-014.

Example 10

Anti-Tumor Efficacy of Anti-Claudin18.2 Antibodies in aPATU8988S-Overexpressing Claudin18.2 Subcutaneous Tumor Model ofPancreatic Cancer

6-7 week old female BALB/c nude mice were injected with 5×10⁶ viablePATU8988S-overexpressing Claudin18.2 cells in 0.1 ml PBS subcutaneouslyinto the right flank. Three days later, mice were randomly sorted intogroups (N=10) and treatment by intraperitoneal injection was initiated(Day 0). Group 1 received PBS control; Group 2 received 200 ug ofisotype antibody control, Group 3 received 200 ug of NBL-014, and Group4 received 200 ug of PC1 antibody. Treatment was administered two timesweekly for 4 weeks.

Body weights were measured twice weekly. Tumor volumes were determinedat different time points using the formula V=½*L×W×W, where L is thelong dimension and W is the short dimension of the xenograft. Any micewith tumors over 2500 mm³ were sacrificed.

As shown in FIG. 18A, the tested antibody NBL-014 and PC1 inhibitedtumor size growth. On day 34 of the study, the mice were sacrified andthe tumors were taken out to measure the weight. Both NBL-014 andin-house PC1 antibody (iMAb362) showed potent anti-tumor efficacy usingtumor weight as the indicator (FIG. 18B).

Example 11

Anti-Tumor Efficacy of Anti-Claudin18.2 Antibodies Iin a NUGC-4Subcutaneous Model Of Gastric Cancer

6-7 week old female BALB/c nude mice were injected with 5×10⁶ viableNUGC-4 cells in 0.1 ml PBS subcutaneously into the right flank. Threedays later, mice were randomly sorted into groups (N=10) and treatmentby intraperitoneal injection was initiated (Day 0). Group 1 received PBScontrol; Group 2 received 200 ug of NBL-014, and Group 3 received 200ugof PC1 antibody The treatment was administered two times weekly for 5weeks.

Body weights were measured twice weekly. Tumor volumes were determinedat different time points using the formula V=½*L×W×W, where L is thelong dimension and W is the short dimension of the xenograft. Any micewith tumors over 2500 mm³ were sacrificed.

As shown in FIG. 19, the tested antibody NBL-014 inhibited tumor growth.On day 35 of the study, both NBL-014 and PC1 showed significantanti-tumor efficacy. NBL-014 exhibited better anti-tumor activity thanin-house PC1 antibody (iMAb362).

Example 12

Anti-Tumor Efficacy of Fc Engineered Anti-Claudin18.2 Antibody in PBMCHumanized NUGC-4 Subcutaneous Model of Gastric Cancer

To investigate the therapeutic potential of an Fc engineeredanti-Clandin18.2 antibody, NOG-MHC I/II-2 KO (NOD.C_(g-B)2m^(em1Tac)Prkdc^(scid) H2-Ab1^(tm1Doi) Il2rg^(tm1Sug)/JicTac) mice from Jacksonlaboratory were chosen to use because of the extended treatment windowwithout GVHD on-set.

Briefly, 6-7 week old female NOG dko mice were inoculated with 5×10⁶viable NUGC-4 cells in 0.1 ml PBS subcutaneously into the right flank.Three days later, peripheral blood mononuclear cells (PBMCs) were thawedand counted. 1×10⁷ PBMC diluted in 300 ul PBS was engrafted to eachmouse through the tail vein. The mice were randomly sorted into groups,and treatment by intraperitoneal injection of antibodies was initiated(Day 0). Group 1 received PBS with PBMC; Group 2 received 200 ug ofNBL-014, which has an engineered human IgG1 Fc.

The treatment was administered two times weekly for 5 weeks. The PBMCengraft was repeated 4 times once a week. The body weights of the micewere measured twice weekly. Tumor volumes were determined at differenttime points using the formula V=½*L×W×W, where L is the long dimensionand W is the short dimension of the xenograft. Any mice with tumors over2500 mm3 were sacrificed.

As Shown in FIGS. 20A and 20B, the group received NBL-014 showedsignificantly reduced tumor volume (FIG. 20A) and tumor weight (FIG.20B) relative to the control group.

1. An isolated fully human anti-human CLDN18.2, wherein the antibodydoes not bind to human CLDN18.1 and the antibody comprises: (a) a heavychain variable region comprising CDR1: SEQ ID NO: 221, CDR2: SEQ ID NO:222, CDR3: SEQ ID NO: 223 and a light chain variable region comprisingCDR1: SEQ ID NO: 224, CDR2: SEQ ID NO: 225, CDR3: SEQ ID NO: 226; (b) aheavy chain variable region comprising CDR1: SEQ ID NO: 227, CDR2: SEQID NO: 228, CDR3: SEQ ID NO: 229 and a light chain variable regioncomprising CDR1: SEQ ID NO: 230, CDR2: SEQ ID NO: 231, CDR3: SEQ ID NO:232; (c) a heavy chain variable region comprising CDR1: SEQ ID NO: 233,CDR2: SEQ ID NO: 234, CDR3: SEQ ID NO: 235 and a light chain variableregion comprising CDR1: SEQ ID NO: 236, CDR2: SEQ ID NO: 237, CDR3: SEQID NO: 238; (d) a heavy chain variable region comprising CDR1: SEQ IDNO: 251, CDR2: SEQ ID NO: 252, CDR3: SEQ ID NO: 253 and a light chainvariable region comprising CDR1: SEQ ID NO: 254, CDR2: SEQ ID NO: 255,CDR3: SEQ ID NO: 256; (e) a heavy chain variable region comprising CDR1:SEQ ID NO: 257, CDR2: SEQ ID NO: 258, CDR3: SEQ ID NO: 259 and a lightchain variable region comprising CDR1: SEQ ID NO: 260, CDR2: SEQ ID NO:261, CDR3: SEQ ID NO: 262; (f) a heavy chain variable region comprisingCDR1: SEQ ID NO: 269, CDR2: SEQ ID NO: 270, CDR3: SEQ ID NO: 271 and alight chain variable region comprising CDR1: SEQ ID NO: 272, CDR2: SEQID NO: 273, CDR3: SEQ ID NO: 274; (g) a heavy chain variable regioncomprising CDR1: SEQ ID NO: 275, CDR2: SEQ ID NO: 276, CDR3: SEQ ID NO:277 and a light chain variable region comprising CDR1: SEQ ID NO: 278,CDR2: SEQ ID NO: 279, CDR3: SEQ ID NO: 280; or (h) a heavy chainvariable region comprising CDR1: SEQ ID NO: 287, CDR2: SEQ ID NO: 288,CDR3: SEQ ID NO: 289 and a light chain variable region comprising CDR1:SEQ ID NO: 290, CDR2: SEQ ID NO: 291, CDR3: SEQ ID NO:
 292. 2.-5.(canceled)
 6. An isolated fully human anti-human CLDN18.2, wherein theantibody does not bind to human CLDN18.1 and the antibody comprises: (a)a heavy chain variable region sequence of SEQ ID NO: 185 and a lightchain variable region sequence of SEQ ID NO: 186; (b) a heavy chainvariable region sequence of SEQ ID NO: 187 and a light chain variableregion sequence of SEQ ID NO: 188; (c) a heavy chain variable regionsequence of SEQ ID NO: 189 and a light chain variable region sequence ofSEQ ID NO: 190; (d) a heavy chain variable region sequence of SEQ ID NO:195 and a light chain variable region sequence of SEQ ID NO: 196; (e) aheavy chain variable region sequence of SEQ ID NO: 197 and a light chainvariable region sequence of SEQ ID NO: 198; (f) a heavy chain variableregion sequence of SEQ ID NO: 201 and a light chain variable regionsequence of SEQ ID NO: 202; (g) a heavy chain variable region sequenceof SEQ ID NO: 203 and a light chain variable region sequence of SEQ IDNO: 204; or (h) a heavy chain variable region sequence of SEQ ID NO: 207and a light chain variable region sequence of SEQ ID NO:
 208. 7. Anisolated Fc-modified anti-human CLDN18.2 comprising VH and VL sequencesselected from: (a) a heavy chain variable region sequence of SEQ ID NO:201 and a light chain variable region sequence of SEQ ID NO: 202; and(b) a heavy chain variable region sequence of SEQ ID NO: 203 and a lightchain variable region sequence of SEQ ID NO: 204 wherein the antibodydoes not bind to CLDN18.1 and further wherein the Fc-modified antibodyis characterized by enhanced ADCC activity relative to the ADCC activityof an antibody comprising the same variable VH and VL regions incombination with a wild-type human IgG1 Fc domain.
 8. An isolatedFc-modified anti-human CLDN18.2 antibody comprising a recombinant heavychain having the amino acid sequence SEQ ID NO :336 and a light chaincomprising the sequence set for in SEQ ID NO:
 337. 9. An isolatedFc-modified anti-human CLDN18.2 antibody comprising a recombinant heavychain having the amino acid sequence SEQ ID NO :338 and a light chaincomprising the sequence set for in SEQ ID NO:
 337. 10. (canceled)